Method for the Production of Polyunsaturated Fatty Acids

Abstract

The present invention relates to a process for producing polyunsaturated fatty acids in an organism by introducing nucleic acids into the organism which code for polypeptides having acyl-CoA:lysophospholipid a cyltransferase activity. Advantageously, these nucleic acid sequences may, if appropriate together with further nucleic acid sequences coding for biosynthesis polypeptides of the fatty acid or lipid metabolism, be expressed in the transgenic organism. The invention furthermore relates to the nucleic acid sequences, to nucleic acid constructs comprising the nucleic acid sequences of the invention, to vectors comprising the nucleic acid sequences and/or the nucleic acid constructs and to transgenic organisms comprising the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors. A further part of the invention relates to oils, lipids and/or fatty acids produced by the process of the invention and to their use.

Description

RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patent application Ser. No. 12/417,171 filed Apr. 2, 2009, which is a divisional of U.S. patent application Ser. No. 10/547,447 filed Aug. 26, 2005, now U.S. Pat. No. 7,537,920, which is a national stage application (under 35 U.S.C. 371) of PCT/EP2004/000771 filed Jan. 29, 2004, which claims benefit to German application 10308836.9 filed Feb. 27, 2003. The entire contents of each of these applications are hereby incorporated by reference herein.

SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing_--12810_--01526. The size of the text file is 273 KB, and the text file was created on Jun. 7, 2013.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to a process for producing polyunsaturated fatty acids in an organism by introducing nucleic acids into said organism which code for polypeptides having acyl-CoA:lysophospholipid-acyltransferase activity. Advantageously, these nucleic acid sequences may, if appropriate together with further nucleic acid sequences coding for biosynthesis polypeptides of the fatty acid or lipid metabolism, be expressed in the transgenic organism.

[0004] The invention furthermore relates to the nucleic acid sequences, to nucleic acid constructs comprising the nucleic acid sequences of the invention, to vectors comprising said nucleic acid sequences and/or said nucleic acid constructs and to transgenic organisms comprising the abovementioned nucleic acid sequences, nucleic acid constructs and/or vectors.

[0005] A further part of the invention relates to oils, lipids and/or fatty acids produced by the process of the invention and to their use.

[0006] Fatty acids and triglycerides have a multiplicity of applications in the food industry, in animal nutrition, in cosmetics and in the pharmacological sector. Depending on whether they are free saturated or unsaturated fatty acids or else triglycerides with an elevated content of saturated or unsaturated fatty acids, they are suitable for very different applications; thus, for example, polyunsaturated fatty acids are added to baby food to improve the nutritional value. Polyunsaturated ω-3-fatty acids and ω-6-fatty acids are, in this connection, an important constituent of animal and human food. Owing to the composition of human food, which is customary today, an addition of polyunsaturated ω-3-fatty acids which are preferably present in fish oils to the food is particularly important. Thus, for example, polyunsaturated fatty acids such as docosahexaenoic acid (=DHA, C22:6.sup.Δ4,7,10,13,16,19) or eisosapentaenoic acid (=EPA, C20:5.sup.Δ5,8,11,14,17) are added to baby food to improve the nutritional value. The unsaturated fatty acid DHA is said to have a positive effect on brain development.

[0007] Hereinbelow, polyunsaturated fatty acids are referred to as PUFA, PUFAs, LCPUFA or LCPUFAs (poly unsaturated fatty acids, PUFA long chain poly unsaturated fatty acids LCPUFA).

[0008] The various fatty acids and triglycerides are obtained, usually in the form of their triacylglycerides (=triglycerides=triglycerols), mainly from microorganisms such as Mortierella or Schizochytrium or from oil-producing plants such as soybean, oilseed rape, algae such as Crypthecodinium or Phaeodactylum and others. However, they may also be obtained from animals such as, for example, fish. The free fatty acids are advantageously prepared by hydrolysis. Higher polyunsaturated fatty acids such as DHA, EPA, arachidonic acid (=ARA, C20:4.sup.Δ5,8,11,14), dihomo-γ-linolenic acid (C20:3.sup.Δ8,11,14) or docosapentaenoic acid (DPA, C22:5.sup.Δ7,10,13,16,19) cannot be isolated from oil crops, such as oilseed rape, soybean, sunflower, safflower or others. Conventional natural sources of these fatty acids are fish such as herring, salmon, sardine, red fish, eel, carp, trout, halibut, mackerel, zander or tuna, or algae.

[0009] Depending on the intended application, preference is given to oils with saturated or unsaturated fatty acids; thus, for example, lipids with unsaturated fatty acids, especially polyunsaturated fatty acids, are preferred in human nutrition. The polyunsaturated ω-3-fatty acids are said to have in this connection a positive effect on the cholesterol level in the blood and thus on the possibility of preventing heart disease. The risk of heart disease, stroke or hypertension may be reduced markedly by adding these ω-3-fatty acids to food. ω-3-fatty acids can also have a positive effect on inflammatory, especially chronically inflammatory, processes in connection with immunological disorders such as rheumatoid arthritis. They are therefore added to food, especially dietetic food, or are applied in medicaments. ω-6-fatty acids such as arachidonic acid tend to have a negative effect on these diseases in connection with said rheumatic disorders, due to our customary foodstuff composition.

[0010] ω-3- and ω-6-fatty acids are precursors of tissue hormones, the "eicosanoides, such as the prostaglandins, which are derived from dihomo-γ-linolenic acid, arachidonic acid and eicosapentaenoic acid, the thromoxanes and leukotrienes which are derived from arachidonic acid and eicosapentaenoic acid. Eicosanoides ("PG₂ series") which are formed from ω-6-fatty acids normally promote inflammatory reactions, while eicosanoides ("PG₃ series") from ω-3-fatty acids have little or no proinflammatory effect.

[0011] Owing to their positive properties, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of fatty acids or triglycerides for the production of oils in various organisms with a modified content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. WO 93/11245 claims a Δ15-desaturase and WO 94/11516 a Δ12-desaturase. Further desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 and Huang et al., Lipids 34, 1999: 649-659. However, the biochemical characterization of the various desaturases has been insufficient to date since the enzymes, being membrane-bound proteins, can be isolated and characterized only with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792). Membrane-bound desaturases are normally characterized by being introduced into a suitable organism which is subsequently studied for enzyme activity by analyzing reactants and products. Δ6-desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No. 5,614,393, WO 96/21022, WO 00/21557 and WO 99/27111, as is the application for production in transgenic organisms, namely in WO 98/46763, WO 98/46764, WO 9846765. The expression of various desaturases such as those in WO 99/64616 or WO 98/46776 and the formation of polyunsaturated fatty acids are also described and claimed in this connection. Regarding the efficacy of desaturase expression and its influence on the formation of polyunsaturated fatty acids, it should be noted that expression of a single desaturase, as described previously, has resulted in only low contents of unsaturated fatty acids/lipids such as, for example, γ-linolenic acid and stearidonic acid. Furthermore, a mixture of ω-3- and ω-6-fatty acids was usually obtained.

[0012] Particularly suitable microorganisms for producing PUFAs are microorganisms such as Thraustochytrium or Schizochytrium strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates, such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor. Strain selection has resulted in the development of a number of mutant strains of the corresponding microorganisms, which produce a series of desirable compounds including PUFAs. However, the mutation and selection of strains with improved production of a particular molecule such as the polyunsaturated fatty acids is a time-consuming and difficult process. Therefore, preference is given, whenever possible, to genetic engineering processes, as described above. However, only limited amounts of the desired polyunsaturated fatty acids such as DPA, EPA or ARA can be produced with the aid of the abovementioned microorganisms, and, depending on the microorganism used, the former are usually obtained as fatty acid mixtures of, for example, EPA, DPA and DHA.

[0013] Alternatively, fine chemicals may be produced advantageously on a large scale via production in plants which are developed so as to produce the abovementioned PUFAs. Plants which are particularly well suited for this purpose are oil crops which contain large amounts of lipid compounds, such as oilseed rape, canola, linseed, soybean, sunflower, borage and evening primrose. However, other crop plants containing oils or lipids and fatty acids are also well suited, as mentioned in the detailed description of the present invention. Conventional breeding has been used to develop a number of mutant plants which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of new plant cultivars with improved production of a particular molecule is a time-consuming and difficult process or even impossible if the compound does not naturally occur in the respective plant, as is the case with polyunsaturated C₁₈-, C₂₀-fatty acids and C₂₂-fatty acids and those having longer carbon chains.

[0014] Owing to the positive properties of unsaturated fatty acids, there has been no lack of attempts in the past to make available these genes which are involved in the synthesis of fatty acids or triglycerides for the production of oils in various plants with a modified content of polyunsaturated fatty acids. Previously, however, it was not possible to produce longer-chain polyunsaturated C₂₀- and/or C₂₂-fatty acids such as EPA or ARA in plants.

[0015] However, in other organisms as well as microorganisms such as algae or fungi too, genetically engineered modifications of the fatty acid metabolic pathway via introducing and expressing, for example, desaturases resulted only in relatively small increases in productivity in these organisms. One reason for this may be the high complexity of the fatty acid metabolism. Thus, incorporation of polyunsaturated fatty acids into membrane lipids and/or into triacylglycerides and their degradation and conversion are very complex and, even now, has still not been fully elucidated and understood biochemically and, especially genetically.

[0016] The biosynthesis of LCPUFAs and incorporation of LCPUFAs into membranes or triacylglycerides are carried out via various metabolic pathways (Abbadi et al. (2001) European Journal of Lipid Science & Technology 103:106-113). In bacteria such as Vibrio and microalgae such as Schizochytrium, malonyl-CoA is converted via a LCPUFA-producing polyketide synthase to give LCPUFAs (Metz et al. (2001) Science 293: 290-293; WO 00/42195; WO 98/27203; WO 98/55625). In microalgae such as Phaeodactylum and mosses such as Physcomitrella, unsaturated fatty acids such as linoleic acid or linolenic acid are converted in the form of their acyl-CoAs in multiple desaturation and elongation steps to give LCPUFAs (Zank et al. (2000) Biochemical Society Transactions 28: 654-658). In mammals, the biosynthesis of DHA includes βoxidation, in addition to desaturation and elongation steps.

[0017] In microorganisms and lower plants, LCPUFAs are present either exclusively in the form of membrane lipids, as is the case in Physcomitrella and Phaeodactylum, or in membrane lipids and triacylglycerides, as is the case in Schizochytrium and Mortierella. Incorporation of LCPUFAs into lipids and oils is catalyzed by various acyltransferases and transacylases. These enzymes are already known to carry out the incorporation of saturated and unsaturated fatty acids [Slabas (2001) J. Plant Physiology 158: 505-513; Frentzen (1998) Fett/Lipid 100: 161-166); Cases et al. (1998) Proc. Nat. Acad. Sci. USA 95: 13018-13023]. The acyltransferases are enzymes of the "Kennedy pathway", which are located on the cytoplasmic side of the membrane system of the endoplasmic reticulum, referred to as "ER" hereinbelow. ER membranes may be isolated experimentally as "microsomal fractions" from various organisms (Knutzon et al. (1995) Plant Physiology 109: 999-1006; Mishra & Kamisaka (2001) Biochemistry 355: 315-322; U.S. Pat. No. 5,968,791). These ER-bound acyltransferases in the microsomal fraction use acyl-CoA as the activated form of fatty acids. Glycerol-3-phosphate acyltransferase, referred to as GPAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-1 position of glycerol 3-phosphate. 1-Acylglycerol-3-phosphate acyltransferase (E.C. 2.3.1.51), also known as lysophosphatidic-acid acyltransferase and referred to as LPAAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-2 position of lysophosphatidic acid, abbreviated as LPA hereinbelow. After dephosphorylation of phosphatidic acid by phosphatidic-acid phosphatase, diacylglycerol acyltransferase, referred to as DAGAT hereinbelow, catalyzes the incorporation of acyl groups at the sn-3 position of diacylglycerols. Apart from these Kennedy pathway enzymes, further enzymes capable of incorporating acyl groups from membrane lipids into triacylglycerides are involved in the incorporation of fatty acids into triacylglycerides, namely phospholipid diacylglycerol acyltransferase, referred to as PDAT hereinbelow, and lysophosphatidylcholine acyltransferase, referred to as LPCAT.

[0018] The enzymic activity of an LPCAT was first described in rats [Land (1960) Journal of Biological Chemistry 235: 2233-2237]. A plastic LPCAT isoform [Akermoun et al. (2000) Biochemical Society Transactions 28: 713-715] and an ER-bound isoform [Tumaney and Rajasekharan (1999) Biochimica et Biophysica Acta 1439: 47-56; Fraser and Stobart, Biochemical Society Transactions (2000) 28: 715-7718] exist in plants. LPCAT is involved in the biosynthesis and transacylation of polyunsaturated fatty acids in animals as well as in plants [Stymne and Stobart (1984) Biochem. J. 223: 305-314; Stymne and Stobart (1987) in `The Biochemistry of Plants: a Comprehensive Treatise`, Vol. 9 (Stumpf, P. K. ed.) pp. 175-214, Academic Press, New York]. An important function of LPCAT or, more generally, of an acyl-CoA:lysophospholipid acyltransferase, referred to as LPLAT hereinbelow, in the ATP-independent synthesis of acyl-CoA from phospholipids has been described by Yamashita et al. (2001; Journal of Biological Chemistry 276: 26745-26752).

[0019] Despite many biochemical data, no genes coding for LPCAT have been identified previously. Genes of various other plant acyltransferases have been isolated and are described in WO 00/18889 (Novel Plant Acyltransferases).

[0020] Higher plants comprise polyunsaturated fatty acids such as linoleic acid (C18:2) and linolenic acid (C18:3). Arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are, as described above, found not at all in the seed oil of higher plants, or only in traces (E. Ucciani: Nouveau Dictionnaire des Huiles Vegetales. Technique & Documentation--Lavoisier, 1995. ISBN: 2-7430-0009-0). It is advantageous to produce LCPUFAs in higher plants, preferably in oil seeds such as oilseed rape, linseed, sunflower and soybean, since large amounts of high-quality LCPUFAs for the food industry, animal nutrition and pharmaceutical purposes may be obtained at low costs in this way. To this end, it is advantageous to introduce into and express in oil seeds genes coding for enzymes of the biosynthesis of LCPUFAs by genetic engineering methods. Said genes encode, for example, Δ6-desaturase, Δ6-elongase, Δ5-desaturase, Δ5-elongase and Δ4-desaturase. These genes may advantageously be isolated from microorganisms, animals and lower plants which produce LCPUFAs and incorporate them in the membranes or triacylglycerides. Thus, Δ6-desaturase genes have already been isolated from the moss Physcomitrella patens and Δ6-elongase genes have already been isolated from P. patens and the nematode C. elegans.

[0021] First transgenic plants which comprise and express genes coding for enzymes of the LCPUFA biosynthesis and produce LCPUFAs have been described for the first time, for example, in DE 102 19 203 (process for the production of polyunsaturated fatty acids in plants). However, these plants produce LCPUFAs in amounts which require further optimization for processing the oils present in said plants.

[0022] In order to enable food and feed to be enriched with these polyunsaturated fatty acids, there is therefore a great need for a simple, inexpensive process for producing said polyunsaturated fatty acids, especially in eukaryotic systems.

BRIEF SUMMARY OF THE INVENTION

[0023] It was therefore the object to develop a process for producing polyunsaturated fatty acids in a eukaryotic organism. This object was achieved by the process according to the invention for producing polyunsaturated fatty acids in an organism, wherein said process comprises the following steps:

[0024] a) introducing into the organism at least one nucleic acid sequence having the sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, which sequence codes for a polypeptide having an acyl-CoA:lysophospholipid-acyltransferase activity; or

[0025] b) introducing into said organism at least one nucleic acid sequence which can be derived, as a result of the degenerated genetic code, from the coding sequence comprised in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or

[0026] c) introducing into said organism at least one derivative of the nucleic acid sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, which code for polypeptides having the amino acid sequence depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and which are at least 40% homologous at the amino acid level to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and have an equivalent acyl-CoA:lysophospholipid-acyltransferase activity, and

[0027] d) culturing and harvesting said organism.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows the amino acid sequence comparison of C. elegans LPLATs (Ce-T06E8.1, SEQ ID NO: 36, and Ce-F59F4.4, SEQ ID NO: 59) with M. musculus LPAAT (Mm-NP061350, SEQ ID NO: 58).

[0029] FIG. 2 shows the fatty acid profiles of transgenic C13ABYS86 S. cerevisiae cells.

[0030] FIG. 3 shows the fatty acid profiles of transgenic C13ABYS86 S. cerevisiae cells.

[0031] FIG. 4 shows the elongation of exogenously applied 18:2.sup.Δ9,12 and 18:3.sup.Δ9,12, 15, following their endogenous Δ6-desaturation (data of FIGS. 2 and 3).

[0032] FIG. 5 shows the fatty acid profiles of transgenic INVSc1 S. cerevisiae cells.

[0033] FIG. 6 shows the fatty acid profiles of transgenic INVSc1 S. cerevisiae cells.

[0034] FIG. 7 shows the acyl-CoA composition of transgenic INVSc1 yeasts transformed with the vectors pESCLeu PpD6Pse1/pYes2 (A) or pESCLeu-PpD6-Pse1/pYes2-T06E8.1 (B).

[0035] FIG. 8 shows the vector map of pSUN3CeLPLAT.

[0036] FIG. 9A shows the vector map of pGPTVLeB4-700+T06E8.1.

[0037] FIG. 9B shows the vector map of pGPTVUSP/OCS-1,2,3 PSE1(Pp)+D6-Des(Pt)+2AT (T06E8-1).

[0038] FIG. 10A shows the biosynthetic pathway of LCPUFAs.

[0039] FIG. 10B shows the biosynthetic pathway of LCPUFAs.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Advantageously, the polyunsaturated fatty acids produced in the process of the invention comprise at least two, advantageously three, double bonds. The fatty acids particularly advantageously comprise four or five double bonds. Fatty acids produced in the process advantageously have 16, 18, 20 or 22 carbon atoms in the fatty acid chain. These fatty acids which have been produced may be produced in said process as a single product or be present in a fatty acid mixture.

[0041] The nucleic acid sequences used in the process of the invention are isolated nucleic acid sequences which code for polypeptides having acyl-CoA:lysophospholipid-acyltransferase activity.

[0042] The polyunsaturated fatty acids produced in the process are advantageously bound in membrane lipids and/or triacylglycerides but may also occur in the organisms as free fatty acids or else bound in the form of other fatty acid esters. In this context, they may be present as "pure products" or else advantageously in the form of mixtures of various fatty acids or mixtures of different glycerides. The various fatty acids bound in the triacylglycerides can be derived here from short-chain fatty acids having from 4 to 6 carbon atoms, medium-chain fatty acids having from 8 to 12 carbon atoms or long-chain fatty acids having from 14 to 24 carbon atoms, with preference being given to the long-chain fatty acids and particular preference being given to the long-chain fatty acids, LCPUFAs, of C₁₈-, C₂₀- and/or C₂₂-fatty acids.

[0043] The process of the invention advantageously produces fatty acid esters with polyunsaturated C₁₆-, C₁₈-, C₂₀- and/or C₂₂-fatty acid molecules, with at least two double bonds being present in the fatty acid ester. These fatty acid molecules preferably comprise three, four or five double bonds and advantageously lead to the synthesis of hexadecadienoic acid, (C16:2.sup.Δ9,12), γ-linolenic acid (=GLA, C18:3.sup.Δ6,9,12), stearidonic acid (=SDA, C18:4.sup.Δ6,9,12,15), dihomo-γ-linolenic acid (=DGLA, 20:3.sup.Δ8,11,14), eicosatetraenoic acid (=ETA, C20:4.sup.Δ5,8,11,14), arachidonic acid (ARA), eicosapentaenoic acid (EPA) or mixtures thereof, preferably EPA and/or ARA.

[0044] The fatty acid esters with polyunsaturated C₁₆-, C₁₈-, C₂₀- and/or C₂₂-fatty acid molecules can be isolated in the form of an oil or lipid, for example in the form of compounds such as sphingolipids, phosphoglycerides, lipids, glycolipids such as glycosphingolipid, phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol, monoacylglycerides, diacylglycerides, triacylglycerides or other fatty acid esters such as the acetyl-coenzyme A esters which comprise the polyunsaturated fatty acids with at least two, preferably three double bonds, from the organisms which have been used for the preparation of the fatty acid esters. In addition to these esters, the polyunsaturated fatty acids are also present in the organisms, advantageously the plants as free fatty acids or bound in other compounds. As a rule, the various abovementioned compounds (fatty acid esters and free fatty acids) are present in the organisms with an approximate distribution of 80 to 90% by weight of triglycerides, 2 to 5% by weight of diglycerides, 5 to 10% by weight of monoglycerides, 1 to 5% by weight of free fatty acids, 2 to 8% by weight of phospholipids, the total of the various compounds amounting to 100% by weight.

[0045] The process according to the invention yields the LCPUFAs produced in a content of at least 3% by weight, advantageously at least 5% by weight, preferably at least 8% by weight, especially preferably at least 10% by weight, most preferably at least 15% by weight, based on the total fatty acids in the transgenic organisms, preferably in a transgenic plant. Since a plurality of reaction steps are performed by the starting compounds hexadecadienoic acid (C16:2), linoleic acid (C18:2) and linolenic acid (C18:3) in the process according to the invention, the end products of the process such as, for example, arachidonic acid (ARA) or eicosapentaenoic acid (EPA) are not obtained as absolutely pure products; minor traces of the precursors are always present in the end product. If, for example, both linoleic acid and linolenic acid are present in the starting organism and the starting plant, the end products such as ARA and EPA are present as mixtures. The precursors should advantageously not amount to more than 20% by weight, preferably not to more than 15% by weight, especially preferably not to more than 10% by weight, most preferably not to more than 5% by weight, based on the amount of the end product in question. Advantageously, only ARA or only EPA, bound or as free acids, are produced as end products in a transgenic plant owing to the process according to the invention. If both compounds (ARA and EPA) are produced simultaneously, they are advantageously produced in a ratio of at least 1:2 (EPA:ARA), advantageously of at least 1:3, preferably 1:4, especially preferably 1:5.

[0046] Owing to the nucleic acid sequences according to the invention, an increase in the yield of polyunsaturated fatty acids of at least 50%, advantageously of at least 80%, especially advantageously of at least 100%, very especially advantageously of at least 150%, in comparison with the nontransgenic starting organism, can be obtained by comparison in GC analysis (see examples).

[0047] Chemically pure polyunsaturated fatty acids or fatty acid compositions can also be synthesized by the processes described above. To this end, the fatty acids or the fatty acid compositions are isolated from the organism, such as the microorganisms or the plants or the culture medium in or on which the organisms have been grown, or from the organism and the culture medium, in the known manner, for example via extraction, distillation, crystallization, chromatography or combinations of these methods. These chemically pure fatty acids or fatty acid compositions are advantageous for applications in the food industry sector, the cosmetics sector and especially the pharmacological industry sector.

[0048] Suitable organisms for the production in the process according to the invention are, in principle, any organisms such as fungi, such as Mortierella or Thraustrochytrium, yeasts such as Saccharomyces or Schizosaccharomyces, mosses such as Physcomitrella or Ceratodon, nonhuman animals such as Caenorhabditis, algae such as Crypthecodinium or Phaeodactylum or plants such as dicotyledonous or monocotyledonous plants. Organisms which are especially advantageously used in the process according to the invention are organisms which belong to the oil-producing organisms, that is to say which are used for the production of oils, such as fungi, such as Mortierella or Thraustochytrium, algae such as Crypthecodinium, Phaeodactylum, or plants, in particular plants, preferably oil crop plants which comprise large amounts of lipid compounds, such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive, sesame, Calendula, Punica, evening primrose, verbascum, thistle, wild roses, hazelnut, almond, macadamia, avocado, bay, pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm, coconut or walnut) or arable crops such as maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa or bushy plants (coffee, cacao, tea), Salix species, and perennial grasses and fodder crops. Preferred plants according to the invention are oil crop plants such as peanut, oilseed rape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, evening primrose, pumpkin/squash, linseed, soybean, borage, trees (oil palm, coconut). Especially preferred are plants which are high in C18:2- and/or C18:3-fatty acids, such as sunflower, safflower, tobacco, verbascum, sesame, cotton, pumpkin/squash, poppy, evening primrose, walnut, linseed, hemp, thistle or safflower. Very especially preferred plants are plants such as safflower, sunflower, poppy, evening primrose, walnut, linseed or hemp.

[0049] It is advantageous to the inventive process described to introduce, in addition to the nucleic acids introduced in steps (a) to (c) of the process, further nucleic acids which encode enzymes of the fatty acid or lipid metabolism.

[0050] In principle, all genes of the fatty acid or lipid metabolism can be used in the process for the production of polyunsaturated fatty acids, advantageously in combination with the inventive acyl-CoA:lysophospholipid acyltransferase. Genes of the fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) are advantageously used in combination with the acyl-CoA:lysophospholipid acyltransferase. Genes selected from the group of the Δ4-desaturases, Δ5-desaturases, Δ6-desaturases, Δ8-desaturases, Δ9-desaturases, Δ12-desaturases, Δ5-elongases, Δ6-elongases or Δ9-elongases are especially preferably used in combination with the acyl-CoA:lysophospholipid acyltransferase in the process of the invention.

[0051] Owing to the enzymatic activity of the nucleic acids used in the process according to the invention which encode polypeptides with acyl-CoA:lysophospholipid acyltransferase activity, advantageously in combination with nucleic acid sequences which encode polypeptides of the fatty acid or lipid metabolism, such as Δ4-, Δ5-, Δ6-, Δ8-desaturase or Δ5-, Δ6- or Δ9-elongase activity, a wide range of polyunsaturated fatty acids can be produced in the process according to the invention. Depending on the choice of the organisms, such as the advantageous plants, used for the process according to the invention, mixtures of the various polyunsaturated fatty acids or individual polyunsaturated fatty acids, such as EPA or ARA, can be produced in free or bound form. Depending on the prevailing fatty acid composition in the starting plant (C18:2- or C18:3-fatty acids), fatty acids which are derived from C18:2-fatty acids, such as GLA, DGLA or ARA, or fatty acids which are derived from C18:3-fatty acids, such as SDA, ETA or EPA, are thus obtained. If only linoleic acid (=LA, C18:2.sup.Δ9,12) is present as unsaturated fatty acid in the plant used for the process, the process can only afford GLA, DGLA and ARA as products, all of which can be present as free fatty acids or in bound form. If only α-linolenic acid (=ALA, C18:3.sup.Δ9,12,15) is present as unsaturated fatty acid in the plant used for the process, as is the case, for example, in linseed, the process can only afford SDA, ETA and EPA as products, all of which can be present as free fatty acids or in bound form, as described above. Owing to the modification of the activity of the enzymes involved in the synthesis, acyl-CoA:lysophospholipid acyltransferase, advantageously in combination with Δ5-, Δ6-desaturase and Δ6-elongase or with Δ5-Δ8-desaturase and Δ9-elongase or in combination with only the first two genes, Δ6-desaturase and Δ6-elongase or Δ8-desaturase and Δ9-elongase, of the synthesis cascade, it is possible to produce, in a targeted fashion, only individual products in the abovementioned organisms, advantageously in the abovementioned plants. Owing to the activity of Δ6-desaturase and Δ6-elongase, for example, GLA and DGLA, or SDA and ETA, are formed, depending on the starting plant and unsaturated fatty acid. DGLA or ETA or mixtures of these are preferably formed. If Δ5-desaturase is additionally introduced into the organisms, advantageously into the plant, ARA or EPA is additionally formed. This also applies to organisms into which Δ8-desaturase and Δ9-elongase have been introduced previously. Advantageously, only ARA or EPA or mixtures of these are synthesized, depending on the fatty acid present in the organism, or in the plant, which acts as starting substance for the synthesis. Since biosynthetic cascades are involved, the end products in question are not present in pure form in the organisms. Small amounts of the precursor compounds are always additionally present in the end product. These small amounts amount to less than 20% by weight, advantageously less than 15% by weight, especially advantageously less than 10% by weight, most advantageously less than 5, 4, 3, 2 or 1% by weight, based on the end products DGLA, ETA or their mixtures, or ARA, EPA or their mixtures.

[0052] To increase the yield in the above-described process for the production of oils and/or triglycerides with an advantageously elevated content of polyunsaturated fatty acids, it is advantageous to increase the amount of starting product for the synthesis of fatty acids; this can be achieved for example by introducing, into the organism, a nucleic acid which encodes a polypeptide with Δ12-desaturase activity.

[0053] This is particularly advantageous in oil-producing organisms such as oilseed rape which are high in oleic acid. Since these organisms are only low in linoleic acid (Mikoklajczak et al., Journal of the American Oil Chemical Society, 38, 1961, 678-681), the use of the abovementioned Δ12-desaturases for producing the starting material linoleic acid is advantageous.

[0054] Nucleic acids used in the process according to the invention are advantageously derived from plants such as algae such as Isochrysis or Crypthecodinium, algae/diatoms such as Phaeodactylum, mosses such as Physcomitrella or Ceratodon, or higher plants such as the Primulaceae such as Aleuritia, Calendula stellata, Osteospermum spinescens or Osteospermum hyoseroides, microorganisms such as fungi, such as Aspergillus, Thraustochytrium, Phytophthora, Entomophthora, Mucor or Mortierella, yeasts or animals such as nematodes such as Caenorhabditis, insects or humans. The nucleic acids are advantageously derived from fungi, animals, or from plants such as algae or mosses, preferably from nematodes such as Caenorhabditis.

[0055] The process according to the invention advantageously employs the abovementioned nucleic acid sequences or their derivatives or homologs which encode polypeptides which retain the enzymatic activity of the proteins encoded by nucleic acid sequences. These sequences, individually or in combination with the nucleic acid sequence which encode acyl-CoA:lysophospholipid acyltransferase, are cloned into expression constructs and used for the introduction into, and expression in, organisms. Owing to their construction, these expression constructs make possible an advantageous optimal synthesis of the polyunsaturated fatty acids produced in the process according to the invention.

[0056] In a preferred embodiment, the process furthermore comprises the step of obtaining a cell or an intact organism which comprises the nucleic acid sequences used in the process, where the cell and/or the organism is transformed with the nucleic acid sequence according to the invention which encodes the acyl-CoA:lysophospholipid acyltransferase, a gene construct or a vector as described above, alone or in combination with further nucleic acid sequences which encode proteins of the fatty acid or lipid metabolism. In a further preferred embodiment, this process furthermore comprises the step of obtaining the fine chemical from the culture. The culture can, for example, take the form of a fermentation culture, for example in the case of the cultivation of microorganisms, such as, for example, Mortierella, Saccharomyces or Thraustochytrium, or a greenhouse- or field-grown culture of a plant. The cell or the organism produced thus is advantageously a cell of an oil-producing organism, such as an oil crop, such as, for example, peanut, oilseed rape, canola, linseed, hemp, soybean, safflower, sunflowers or borage.

[0057] In the case of plant cells, plant tissue or plant organs, "growing" is understood as meaning, for example, the cultivation on or in a nutrient medium, or of the intact plant on or in a substrate, for example in a hydroponic culture, potting compost or on arable land.

[0058] For the purposes of the invention, "transgenic" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette (=gene construct) or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassette or vector according to the invention, all those constructions brought about by recombinant methods in which either

[0059] a) the nucleic acid sequence according to the invention, or

[0060] b) a genetic control sequence which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or

[0061] c) (a) and (b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original organism or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette--for example the naturally occurring combination of the natural promoter of the inventive nucleic acid sequence with the corresponding acyl-CoA:lysophospholipid acyltransferase gene--becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

[0062] A transgenic organism or transgenic plant for the purposes of the invention is understood as meaning, as above, that the nucleic acids used in the process are not at their natural locus in the genome of an organism, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention are at their natural position in the genome of an organism, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic organisms are fungi such as Mortierella or plants such as the oil crops.

[0063] Organisms or host organisms for the nucleic acids, the expression cassette or the vector used in the process according to the invention are, in principle, advantageously all organisms which are capable of synthesizing fatty acids, specifically unsaturated fatty acids, and/or which are suitable for the expression of recombinant genes. Examples which may be mentioned are plants such as Arabidopsis, Asteraceae such as Calendula or crop plants such as soybean, peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) or cacao bean, microorganisms, such as fungi, for example the genus Mortierella, Saprolegnia, or Pythium, bacteria, such as the genus Escherichia, yeasts, such as the genus Saccharomyces, cyanobacteria, ciliates, algae or protozoans such as dinoflagellates, such as Crypthecodinium. Preferred organisms are those which are naturally capable of synthesizing substantial amounts of oil, such as fungi, such as Mortierella alpina, Pythium insidiosum, or plants such as soybean, oilseed rape, coconut, oil palm, safflower, flax, hemp, castor-oil plant, Calendula, peanut, cacao bean or sunflower, or yeasts such as Saccharomyces cerevisiae with soybean, flax, oilseed rape, safflower, sunflower, Calendula, Mortierella or Saccharomyces cerevisiae being especially preferred. In principle, host organisms are, in addition to the abovementioned transgenic organisms, also transgenic animals, advantageously nonhuman animals, for example C. elegans.

[0064] Further utilizable host cells are detailed in: Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0065] Expression strains which can be used, for example those with a lower protease activity, are described in: Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128.

[0066] These include plant cells and certain tissues, organs and parts of plants in all their phenotypic forms such as anthers, fibers, root hairs, stalks, embryos, calli, cotyledons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures which are derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant.

[0067] Transgenic plants which comprise the polyunsaturated fatty acids synthesized in the process according to the invention can advantageously be marketed directly without there being any need for the oils, lipids or fatty acids synthesized to be isolated. Plants for the process according to the invention are listed as meaning intact plants and all plant parts, plant organs or plant parts such as leaf, stem, seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos, calli, cotyledons, petioles, harvested material, plant tissue, reproductive tissue and cell cultures which are derived from the actual transgenic plant and/or can be used for bringing about the transgenic plant. In this context, the seed comprises all parts of the seed such as the seed coats, epidermal cells, seed cells, endosperm or embryonic tissue. However, the compounds produced in the process according to the invention can also be isolated from the organisms, advantageously plants, in the form of their oils, fat, lipids and/or free fatty acids. Polyunsaturated fatty acids produced by this process can be obtained by harvesting the organisms, either from the crop in which they grow, or from the field. This can be done via pressing or extraction of the plant parts, preferably the plant seeds. In this context, the oils, fats, lipids and/or free fatty acids can be obtained by what is known as cold-beating or cold-pressing without applying heat by pressing. To allow for greater ease of disruption of the plant parts, specifically the seeds, they are previously comminuted, steamed or roasted. The seeds which have been pretreated in this manner can subsequently be pressed or extracted with solvents such as warm hexane. The solvent is subsequently removed. In the case of microorganisms, the latter are, after harvesting, for example extracted directly without further processing steps or else, after disruption, extracted via various methods with which the skilled worker is familiar. In this manner, more than 96% of the compounds produced in the process can be isolated. Thereafter, the resulting products are processed further, i.e. refined. In this process, substances such as the plant mucilages and suspended matter are first removed. What is known as desliming can be effected enzymatically or, for example, chemico-physically by addition of acid such as phosphoric acid. Thereafter, the free fatty acids are removed by treatment with a base, for example sodium hydroxide solution. The resulting product is washed thoroughly with water to remove the alkali remaining in the product and then dried. To remove the pigments remaining in the product, the products are subjected to bleaching, for example using filler's earth or active charcoal. At the end, the product is deodorized, for example using steam.

[0068] The PUFAs or LCPUFAs produced by this process are advantageously C₁₈-, C₂₀- or C₂₂-fatty acid molecules with at least two double bonds in the fatty acid molecule, preferably three, four, five or six double bonds. These C₁₈-, C₂₀- or C₂₂-fatty acid molecules can be isolated from the organism in the form of an oil, a lipid or a free fatty acid. Suitable organisms are, for example, those mentioned above. Preferred organisms are transgenic plants.

[0069] One embodiment of the invention is therefore oils, lipids or fatty acids or fractions thereof which have been produced by the above-described process, especially preferably oil, lipid or a fatty acid composition comprising PUFAs and being derived from transgenic plants.

[0070] A further embodiment according to the invention is the use of the oil, lipid, the fatty acids and/or the fatty acid composition in feedstuffs, foodstuffs, cosmetics or pharmaceuticals.

[0071] The term "oil", "lipid" or "fat" is understood as meaning a fatty acid mixture comprising unsaturated or saturated, preferably esterified, fatty acid(s). The oil, lipid or fat is preferably high in polyunsaturated free or, advantageously, esterified fatty acid(s), in particular linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid. The content of unsaturated esterified fatty acids preferably amounts to approximately 30%, a content of 50% is more preferred, a content of 60%, 70%, 80% or more is even more preferred. For the analysis, the fatty acid content can, for example, be determined by gas chromatography after converting the fatty acids into the methyl esters by transesterification. The oil, lipid or fat can comprise various other saturated or unsaturated fatty acids, for example calendulic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and the like. The content of the various fatty acids in the oil or fat can vary, in particular depending on the starting organism.

[0072] The polyunsaturated fatty acids with advantageously at least two double bonds which are produced in the process are, as described above, for example sphingolipids, phosphoglycerides, lipids, glycolipids, phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol or other fatty acid esters.

[0073] Starting from the polyunsaturated fatty acids with advantageously at least two double bonds, which acids have been prepared in the process according to the invention, the polyunsaturated fatty acids which are present can be liberated for example via treatment with alkali, for example aqueous KOH or NaOH, or acid hydrolysis, advantageously in the presence of an alcohol such as methanol or ethanol, or via enzymatic cleavage, and isolated via, for example, phase separation and subsequent acidification via, for example, H₂SO₄. The fatty acids can also be liberated directly without the above-described processing step.

[0074] After their introduction into an organism, advantageously a plant cell or plant, the nucleic acids used in the process can either be present on a separate plasmid or integrated into the genome of the host cell. In the case of integration into the genome, integration can be random or else be effected by recombination such that the native gene is replaced by the copy introduced, whereby the production of the desired compound by the cell is modulated, or by the use of a gene in trans, so that the gene is linked operably with a functional expression unit which comprises at least one sequence which ensures the expression of a gene and at least one sequence which ensures the polyadenylation of a functionally transcribed gene. The nucleic acids are advantageously introduced into the organisms via multiexpression cassettes or constructs for multiparallel expression, advantageously into the plants for the multiparallel seed-specific expression of genes.

[0075] Mosses and algae are the only known plant systems which produce substantial amounts of polyunsaturated fatty acids such as arachidonic acid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA). Mosses comprise PUFAs in membrane lipids, while algae, organisms which are related to algae and a few fungi also accumulate substantial amounts of PUFAs in the triacylglycerol fraction. This is why nucleic acid molecules which are isolated from such strains which also accumulate PUFAs in the triacylglycerol fraction are particularly advantageous for the process according to the invention and thus for the modification of the lipid and PUFA production system in a host, in particular plants such as oil crops, for example oilseed rape, canola, linseed, hemp, soybeans, sunflowers and borage. They can therefore be used advantageously in the process according to the invention.

[0076] Substrates of the acyl-CoA:lysophospholipid acyltransferase(s) which are advantageously used are C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids.

[0077] To produce the long-chain PUFAs according to the invention, the polyunsaturated C₁₆- or C₁₈-fatty acids must first be desaturated by the enzymatic activity of a desaturase and subsequently be elongated by at least two carbon atoms via an elongase. After one elongation cycle, this enzyme activity gives C₁₈- or C₂₀-fatty acids and after two or three elongation cycles C₂₂- or C₂4-fatty acids. The activity of the desaturases and elongases used in the process according to the invention preferably leads to C₁₈-, C₂₀- and/or C₂₂-fatty acids, advantageously with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds, especially preferably to give C₂₀- and/or C₂₂-fatty acids with at least two double bonds in the fatty acid molecule, preferably with three, four or five double bonds in the molecule. After a first desaturation and the elongation have taken place, further desaturation steps such as, for example, one in the Δ5 position may take place. Products of the process according to the invention which are especially preferred are dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoic acid. The C₁₈-fatty acids with at least two double bonds in the fatty acid can be elongated by the enzymatic activity according to the invention in the form of the free fatty acid or in the form of the esters, such as phospholipids, glycolipids, sphingolipids, phosphoglycerides, monoacylglycerol, diacylglycerol or triacylglycerol.

[0078] The preferred biosynthesis site of the fatty acids, oils, lipids or fats in the plants which are advantageously used is, for example, in general the seed or cell strata of the seed, so that seed-specific expression of the nucleic acids used in the process makes sense. However, it is obvious that the biosynthesis of fatty acids, oils or lipids need not be limited to the seed tissue, but can also take place in a tissue-specific manner in all the other parts of the plant, for example in epidermal cells or in the tubers.

[0079] If microorganisms such as yeasts, such as Saccharomyces or Schizosaccharomyces, fungi such as Mortierella, Aspergillus, Phytophtora, Entomophthora, Mucor or Thraustochytrium, algae such as Isochrysis, Phaeodactylum or Crypthecodinium are used as organisms in the process according to the invention, these organisms are advantageously grown in fermentation cultures.

[0080] Owing to the use of the nucleic acids according to the invention which encode acyl-CoA:lysophospholipid acyltransferase(s), the polyunsaturated fatty acids produced in the process can be increased by at least 10%, preferably by at least 15%, especially preferably by at least 20%, very especially preferably by at least 50% in comparison with the wild type of the organisms which do not comprise the nucleic acids recombinantly.

[0081] In principle, the polyunsaturated fatty acids produced by the process according to the invention in the organisms used in the process can be increased in two different ways. Advantageously, the pool of free polyunsaturated fatty acids and/or the content of the esterified polyunsaturated fatty acids produced via the process can be enlarged. Advantageously, the pool of esterified polyunsaturated fatty acids in the transgenic organisms is enlarged by the process according to the invention.

[0082] If microorganisms are used as organisms in the process according to the invention, they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while passing in oxygen. The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semicontinuously or continuously. The polyunsaturated fatty acids produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. To this end, the organisms can advantageously be disrupted beforehand.

[0083] If the host organisms are microorganisms, the process according to the invention is advantageously carried out at a temperature of between 0° C. and 95° C., preferably between 10° C. and 85° C., especially preferably between 15° C. and 75° C., very especially preferably between 15° C. and 45° C.

[0084] In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

[0085] The process according to the invention can be operated batchwise, semibatchwise or continuously. An overview over known cultivation methods can be found in the textbook by Chmiel (Bioprozeβtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

[0086] The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

[0087] As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements.

[0088] Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of very good carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar raffination. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanol, and/or organic acids such as, for example, acetic acid and/or lactic acid.

[0089] Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

[0090] Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0091] Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine.

[0092] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

[0093] Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

[0094] The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

[0095] All media components are sterilized, either by heat (20 min at 1.5 bar and 121° C.) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

[0096] The culture temperature is normally between 15° C. and 45° C., preferably at from 25° C. to 40° C., and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20° to 45° C. and preferably 25° C. to 40° C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.

[0097] The fermentation broths obtained in this way, in particular those containing polyunsaturated fatty acids, usually contain a dry mass of from 7.5 to 25% by weight.

[0098] The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.

[0099] However, the fermentation broth can also be thickened or concentrated without separating the cells, using known methods such as, for example, with the aid of a rotary evaporator, thin-film evaporator, falling-film evaporator, by reverse osmosis or by nanofiltration. Finally, this concentrated fermentation broth can be processed to obtain the fatty acids present therein.

[0100] The fatty acids obtained in the process are also suitable as starting material for the chemical synthesis of further products of interest. For example, they can be used in combination with one another or alone for the preparation of pharmaceuticals, foodstuffs, animal feeds or cosmetics.

[0101] The invention furthermore relates to isolated nucleic acid sequences coding for polypeptides having acyl-CoA:lysophospholipid acyltransferase activity wherein the acyl-CoA:lysophospholipid acyltransferases encoded by said nucleic acid sequences specifically convert C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids having at least one double bond in the fatty acid molecule.

[0102] Advantageous isolated nucleic acid sequences are sequences selected from the group consisting of:

[0103] a) a nucleic acid sequence having the sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,

[0104] b) nucleic acid sequences which can be derived from the coding sequence comprised in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 as a result of the degenerated genetic code

[0105] c) derivatives of the nucleic acid sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 which code for polypeptides having the amino acid sequence depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and are at least 40% homologous at the amino acid level to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and have an acyl-CoA:lysophospholipid-acyltransferase activity.

[0106] The abovementioned nucleic acid sequences are advantageously derived from a eukaryotic organism.

[0107] The nucleic acid sequences used in the process which code for proteins with acyl-CoA:lysophospholipid acyltransferase activity or for proteins of the fatty acid or lipid metabolism are advantageously introduced in an expression cassette (=nucleic acid construct) which makes possible the expression of the nucleic acids in an organism, advantageously a plant or a microorganism.

[0108] To introduce the nucleic acids used in the process, the latter are advantageously amplified and ligated in the known manner. Preferably, a procedure following the protocol for Pfu DNA polymerase or a Pfu/Taq DNA polymerase mixture is followed. The primers are selected taking into consideration the sequence to be amplified. The primers should advantageously be chosen in such a way that the amplificate comprises the entire codogenic sequence from the start codon to the stop codon. After the amplification, the amplificate is expediently analyzed. For example, a gel-electrophoretic separation can be carried out, which is followed by a quantitative and a qualitative analysis. Thereafter, the amplificate can be purified following a standard protocol (for example Qiagen). An aliquot of the purified amplificate is then available for the subsequent cloning step. Suitable cloning vectors are generally known to the skilled worker. These include, in particular, vectors which are capable of replication in microbial systems, that is to say mainly vectors which ensure efficient cloning in yeasts or fungi and which make possible the stable transformation of plants. Those which must be mentioned in particular are various binary and cointegrated vector systems which are suitable for the T-DNA-mediated transformation. Such vector systems are, as a rule, characterized in that they comprise at least the vir genes required for the Agrobacterium-mediated transformation and the T-DNA-delimiting sequences (T-DNA border). These vector systems advantageously also comprise further cis-regulatory regions such as promoters and terminator sequences and/or selection markers, by means of which suitably transformed organisms can be identified. While in the case of cointegrated vector systems vir genes and T-DNA sequences are arranged on the same vector, binary systems are based on at least two vectors, one of which bears vir genes, but no T-DNA, while a second one bears T-DNA, but no vir gene. Owing to this fact, the last-mentioned vectors are relatively small, easy to manipulate and to replicate both in E. coli and in Agrobacterium. These binary vectors include vectors from the series pBIB-HYG, pPZP, pBecks, pGreen. In accordance with the invention, pBin19, pBI101, pBinAR, pGPTV and pCAMBIA are used by preference. An overview of the binary vectors and their use is found in Hellens et al, Trends in Plant Science (2000) 5, 446-451. In order to prepare the vectors, the vectors can first be linearized with restriction endonuclease(s) and then modified enzymatically in a suitable manner. Thereafter, the vector is purified, and an aliquot is employed for the cloning step. In the cloning step, the enzymatically cleaved and, if appropriate, purified amplificate is ligated with vector fragments which have been prepared in a similar manner, using ligase. In this context, a particular nucleic acid construct, or vector or plasmid construct, can have one or else more than one codogenic gene segment. The codogenic gene segments in these constructs are preferably linked operably with regulatory sequences. The regulatory sequences include, in particular, plant sequences such as the above-described promoters and terminator sequences. The constructs can advantageously be stably propagated in microorganisms, in particular in Escherichia coli and Agrobacterium tumefaciens, under selective conditions and make possible the transfer of heterologous DNA into plants or microorganisms.

[0109] The nucleic acids used in the process, the inventive nucleic acids and nucleic acid constructs, can be introduced into organisms such as microorganisms or advantageously plants, advantageously using cloning vectors, and thus be used in the transformation of plants such as those which are published and cited in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Fla.), Chapter 6/7, p. 71-119 (1993); F. F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225. Thus, the nucleic acids, the inventive nucleic acids and nucleic acid constructs, and/or vectors used in the process can be used for the recombinant modification of a broad spectrum of organisms, advantageously plants, so that the latter become better and/or more efficient PUFA producers.

[0110] A series of mechanisms exists by which the modification of an acyl-CoA:lysophospholipid acyltransferase protein can influence directly the yield, production and/or production efficiency of a fine chemical from an oil crop plant or a microorganism, owing to a modified protein. The number or activity of the acyl-CoA:lysophospholipid acyltransferase protein or gene and also of gene combinations of acyl-CoA:lysophospholipid acyltransferases, desaturases and/or elongases may have increased, so that greater amounts of the compounds produced are produced de novo, since the organisms lacked this activity and ability to biosynthesize prior to introduction of the corresponding gene(s). This applies analogously to the combination with further desaturases or elongases or further enzymes of the fatty acid and lipid metabolism. The use of various divergent sequences, i.e. sequences which differ at the DNA sequence level, may also be advantageous in this context, or else the use of promoters for gene expression which makes possible a different gene expression in the course of time, for example as a function of the degree of maturity of a seed or an oil-storing tissue.

[0111] Owing to the introduction of one or more acyl-CoA:lysophospholipid acyltransferase, desaturase and/or elongase genes into an organism, alone or in combination with other genes in a cell, it is not only possible to increase biosynthesis flux towards the end product, but also to increase, or to create de novo the corresponding triacylglycerol composition. Likewise, the number or activity of other genes which are involved in the import of nutrients which are required for the biosynthesis of one or more fine chemicals (e.g. fatty acids, polar and/or neutral lipids), can be increased, so that the concentration of these precursors, cofactors or intermediates within the cells or within the storage compartment is increased, whereby the ability of the cells to produce PUFAs as described below is enhanced further. Fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more acyl-CoA:lysophospholipid acyltransferases, desaturases and/or elongases which are involved in the biosynthesis of these compounds, or by destroying the activity of one or more desaturases which are involved in the degradation of these compounds, an enhanced yield, production and/or efficiency of production of fatty acid and lipid molecules in organisms, advantageously in plants, is made possible.

[0112] The isolated nucleic acid molecules used in the process according to the invention encode proteins or parts of these, where the proteins or the individual protein or parts thereof comprise(s) an amino acid sequence with sufficient homology to an amino acid sequence which is shown in the sequence SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, so that the protein or part thereof retains an acyl-CoA:lysophospholipid acyltransferase activity. The protein or part thereof which is encoded by the nucleic acid molecule preferably retains its essential enzymatic activity and the ability to participate in the metabolism of compounds required for the synthesis of cell membranes or lipid bodies in organisms, advantageously in plants, or in the transport of molecules across these membranes. Advantageously, the protein encoded by the nucleic acid molecules is at least approximately 40%, preferably at least approximately 60% and more preferably at least approximately 70%, 80% or 90% and most preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. Advantageous embodiments of the inventive amino acid sequence of the sequence SEQ ID NO: 2 are amino acid sequences which have a valine residue instead of the methionine at position 30 of SEQ ID NO: 2 or have a glycine residue instead of the serine at position 100 or have a serine residue instead of the phenylalanine at position 170. These are indicated in SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

[0113] Essential enzymatic activity of the acyl-CoA:lysophospholipid acyltransferases used is understood as meaning that they retain at least an enzymatic activity of at least 10%, preferably 20%, especially preferably 30% and very especially 40% in comparison with the proteins/enzymes encoded by the sequence with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 and their derivatives and can thus participate in the metabolism of compounds required for the synthesis of fatty acids in an organism, advantageously a plant cell, or in the transport of molecules across membranes, meaning desaturated C₁₆-, C₁₈- or C₂₀-24-carbon chains with double bonds at at least two, advantageously three, four or five positions.

[0114] Nucleic acids which can advantageously be used in the process are derived from fungi or plants such as algae or mosses, such as the genera Physcomitrella, Thraustochytrium, Phytophthora, Ceratodon, Isochrysis, Aleurita, Muscarioides, Mortierella, Borago, Phaeodactylum, Crypthecodinium or from nematodes such as Caenorhabditis, specifically from the genera and species Physcomitrella patens, Phytophtora infestans, Ceratodon purpureus, Isochrysis galbana, Aleurita farinosa, Muscarioides viallii, Mortierella alpina, Borago officinalis, Phaeodactylum tricornutum, or especially advantageously from Caenorhabditis elegans.

[0115] Alternatively, the isolated nucleotide sequences used may encode acyl-CoA:lysophospholipid acyltransferases which hybridize with a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, for example under stringent conditions.

[0116] The nucleic acid sequences used in the process are advantageously introduced into an expression cassette which makes possible the expression of the nucleic acids in organisms such as microorganisms or plants.

[0117] In doing so, the nucleic acid sequences which encode the acyl-CoA:lysophospholipid acyltransferases of the invention, the desaturases used and/or the elongases are linked operably with one or more regulatory signals, advantageously for enhancing gene expression. These regulatory sequences are intended to make possible the specific expression of the genes and proteins. Depending on the host organism, this may mean, for example, that the gene is expressed and/or overexpressed only after induction has taken place, or else that it expresses and/or overexpresses immediately. For example, these regulatory sequences take the form of sequences to which inductors or repressors bind, thus controlling the expression of the nucleic acid. In addition to these novel regulatory sequences, or instead of these sequences, the natural regulation of these sequences may still be present before the actual structural genes and, if appropriate, may have been genetically modified in such a way that natural regulation has been eliminated and expression of the genes has been enhanced. However, the expression cassette (=expression construct=gene construct) can also be simpler in construction, that is to say no additional regulatory signals have been inserted before the nucleic acid sequence or its derivatives, and the natural promoter together with its regulation was not removed. Instead, the natural regulatory sequence has been mutated in such a way that regulation no longer takes place and/or gene expression is enhanced. These modified promoters can also be positioned on their own before the natural gene in the form of part-sequences (=promoter with parts of the nucleic acid sequences used in accordance with the invention) in order to enhance the activity. Moreover, the gene construct may advantageously also comprise one or more what are known as enhancer sequences in operable linkage with the promoter, which make possible an enhanced expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminator sequences, may also be inserted at the 3' end of the DNA sequences. The acyl-CoA:lysophospholipid acyltransferase genes and the advantageously used Δ4-desaturase, Δ5-desaturase, Δ6-desaturase and/or 48-desaturase genes and/or Δ5-elongase, Δ6-elongase and/or Δ9-elongase genes may be present in one or more copies in the expression cassette (=gene construct). Preferably, only one copy of the genes is present in each expression cassette. This gene construct or the gene constructs can be expressed together in the host organism. In this context, the gene construct(s) can be inserted in one or more vectors and be present in the cell in free form, or else be inserted in the genome. It is advantageous for the insertion of further genes in the host genome when the genes to be expressed are present together in one gene construct.

[0118] In this context, the regulatory sequences or factors can, as described above, preferably have a positive effect on the gene expression of the genes introduced, thus enhancing it. Thus, an enhancement of the regulatory elements, advantageously at the transcriptional level, may take place by using strong transcription signals such as promoters and/or enhancers. In addition, however, enhanced translation is also possible, for example by improving the stability of the mRNA.

[0119] A further embodiment of the invention is one or more gene constructs which comprise one or more sequences which are defined by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or its derivatives and which encode polypeptides as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. The abovementioned acyl-CoA:lysophospholipid acyltransferases lead advantageously to an exchange of fatty acids between the mono-, di- and/or triglyceride pool of the cell and the CoA-fatty acid ester pool, the substrate advantageously having one, two, three, four or five double bonds and advantageously 16, 18, 20, 22 or 24 carbon atoms in the fatty acid molecule. The same applies to their homologs, derivatives or analogs, which are linked operably with one or more regulatory signals, advantageously for enhancing gene expression.

[0120] Advantageous regulatory sequences for the novel process are present for example in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6, λ-PR or Δ-PL promoter and are advantageously employed in Gram-negative bacteria. Further advantageous regulatory sequences are, for example, present in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plant promoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS, lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter. Advantageous in this context are also inducible promoters, such as the promoters described in EP-A-0 388 186 (benzenesulfonamide-inducible), Plant J. 2, 1992:397-404 (Gatz et al., tetracycline-inducible), EP-A-0 335 528 (abscissic acid-inducible) or WO 93/21334 (ethanol- or cyclohexenol-inducible) promoters. Further suitable plant promoters are the cytosolic FBPase promoter or the ST-LSI promoter of potato (Stockhaus et al., EMBO J. 8, 1989, 2445), the glycine max phosphoribosylpyrophosphate amidotransferase promoter (Genbank Accession No. U87999) or the node-specific promoter described in EP-A-0 249 676. Especially advantageous promoters are promoters which make possible the expression in tissues which are involved in the biosynthesis of fatty acids. Very especially advantageous are seed-specific promoters, such as the USP promoter as described, but also other promoters such as the LeB4, DC3, phaseolin or napin promoter. Further especially advantageous promoters are seed-specific promoters which can be used for monocotyledonous or dicotyledonous plants and which are described in U.S. Pat. No. 5,608,152 (oilseed rape napin promoter), WO 98/45461 (Arabidopsis oleosin promoter), U.S. Pat. No. 5,504,200 (Phaseolus vulgaris phaseolin promoter), WO 91/13980 (Brassica Bce4 promoter), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4 promoter from a legume), these promoters being suitable for dicots. Examples of promoters which are suitable for monocots are the barley Ipt-2 or Ipt-1 promoter (WO 95/15389 and WO 95/23230), the barley hordein promoter and other suitable promoters described in WO 99/16890.

[0121] In principle, it is possible to use all natural promoters together with their regulatory sequences, such as those mentioned above, for the novel process. It is also possible and advantageous to use synthetic promoters, either in addition or alone, in particular when they mediate seed-specific expression, such as those described in WO 99/16890.

[0122] In order to achieve a particularly high PUFA content, especially in transgenic plants, the PUFA biosynthesis genes should advantageously be expressed in oil crops in a seed-specific manner. To this end, seed-specific promoters can be used, or those promoters which are active in the embryo and/or in the endosperm. In principle, seed-specific promoters can be isolated both from dicotyledonous and from monocotyledonous plants. Preferred promoters are listed hereinbelow: USP (=unknown seed protein) and vicilin (Vicia faba) [Baumlein et al., Mol. Gen. Genet., 1991, 225(3)], napin (oilseed rape) [U.S. Pat. No. 5,608,152], acyl carrier protein (oilseed rape) [U.S. Pat. No. 5,315,001 and WO 92/18634], oleosin (Arabidopsis thaliana) [WO 98/45461 and WO 93/20216], phaseolin (Phaseolus vulgaris) [U.S. Pat. No. 5,504,200], Bce4 [WO 91/13980], legumines B4 (LegB4 promoter) [Baumlein et al., Plant J., 2, 2, 1992], Lpt2 and Ipt1 (barley) [WO 95/15389 and WO 95/23230], seed-specific promoters from rice, maize and wheat [WO 99/16890], Amy32b, Amy 6-6 and aleurain [U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin (soybean) [EP 571 741], phosphoenol pyruvate carboxylase (soybean) [JP 06/62870], ADR12-2 (soybean) [WO 98/08962], isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase (barley) [EP 781 849].

[0123] Plant gene expression can also be facilitated via a chemically inducible promoter (see review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired that gene expression should take place in a time-specific manner. Examples of such promoters are a salicylic-acid-inducible promoter (WO 95/19443), a tetracycline-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.

[0124] To ensure the stable integration of the biosynthesis genes into the transgenic plant over a plurality of generations, each of the nucleic acids which encode acyl-CoA:lysophospholipid acyltransferase, the advantageous Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ8-desaturase and/or Δ5-elongase, Δ6-elongase and/or Δ9-elongase and which are used in the process should be expressed under the control of a separate promoter, preferably a promoter which differs from the other promoters, since repeating sequence motifs can lead to instability of the T-DNA, or to recombination events. In this context, the expression cassette is advantageously constructed in such a way that a promoter is followed by a suitable cleavage site, advantageously in a polylinker, for insertion of the nucleic acid to be expressed and, if appropriate, a terminator sequence is positioned behind the polylinker. This sequence is repeated several times, preferably three, four or five times, so that up to five genes can be combined in one construct and introduced into the transgenic plant in order to be expressed. Advantageously, the sequence is repeated up to three times. To express the nucleic acid sequences, the latter are inserted behind the promoter via the suitable cleavage site, for example in the polylinker. Advantageously, each nucleic acid sequence has its own promoter and, if appropriate, its own terminator sequence. However, it is also possible to insert a plurality of nucleic acid sequences behind a promoter and, if appropriate, before a terminator sequence. Here, the insertion site, or the sequence, of the inserted nucleic acids in the expression cassette is not of critical importance, that is to say a nucleic acid sequence can be inserted at the first or last position in the cassette without its expression being substantially influenced thereby. Advantageously, different promoters such as, for example, the USP, LegB4 or DC3 promoter, and different terminator sequences can be used in the expression cassette. However, it is also possible to use only one type of promoter in the cassette. This, however, may lead to undesired recombination events.

[0125] As described above, the transcription of the genes which have been introduced should advantageously be terminated by suitable terminator sequences at the 3' end of the biosynthesis genes which have been introduced (behind the stop codon). An example of a sequence which can be used in this context is the OCS 1 terminator sequence. As is the case with the promoters, different terminator sequences should be used for each gene.

[0126] As described above, the gene construct can also comprise further genes to be introduced into the organisms. It is possible and advantageous to introduce into the host organisms, and to express therein, regulatory genes such as genes for inductors, repressors or enzymes which, owing to their enzyme activity, engage in the regulation of one or more genes of a biosynthesis pathway. These genes can be of heterologous or of homologous origin. Moreover, further biosynthesis genes of the fatty acid or lipid metabolism can advantageously be present in the nucleic acid construct, or gene construct; however, these genes can also be positioned on one or more further nucleic acid constructs. Biosynthesis genes of the fatty acid or lipid metabolism which are preferably used are a gene selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases or fatty acid elongase(s) or combinations thereof. Especially advantageous nucleic acid sequences are biosynthesis genes of the fatty acid or lipid metabolism selected from the group of the Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ8-desaturase, Δ9-desaturase, 2-desaturase, Δ5-elongase, Δ6-elongase or Δ9-elongase.

[0127] In this context, the abovementioned desaturases can be cloned into expression cassettes of the invention in combination with other elongases and desaturases and used for transforming plants with the aid of Agrobacterium.

[0128] Here, the regulatory sequences or factors can, as described above, preferably have a positive effect on, and thus enhance, the expression of the genes which have been introduced. Thus, enhancement of the regulatory elements can advantageously take place at the transcriptional level by using strong transcription signals such as promoters and/or enhancers. However, an enhanced translation is also possible, for example by improving the stability of the mRNA. In principle, the expression cassettes can be used directly for introduction into the plants or else be introduced into a vector.

[0129] These advantageous vectors, preferably expression vectors, comprise the nucleic acids which encode acyl-CoA:lysophospholipid acyltransferases and which are used in the process, or else a nucleic acid construct which comprises the nucleic acid used either alone or in combination with further biosynthesis genes of the fatty acid or lipid metabolism such as Δ4-desaturase, Δ5-desaturase, Δ6-desaturase, Δ8-desaturase, Δ9-desaturase, 2-desaturase, Δ5-elongase, Δ6-elongase and/or Δ9-elongase. As used in the present context, the term "vector" refers to a nucleic acid molecule which is capable of transporting another nucleic acid to which it is bound. One type of vector is a "plasmid", a circular double-stranded DNA loop into which additional DNA segments can be ligated. A further type of vector is a viral vector, it being possible for additional DNA segments to be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they have been introduced (for example bacterial vectors with bacterial replication origin). Other vectors are advantageously integrated into the genome of a host cell when they are introduced into the host cell, and thus replicate together with the host genome. Moreover, certain vectors can govern the expression of genes with which they are in operable linkage. These vectors are referred to in the present context as "expression vectors". Usually, expression vectors which are suitable for DNA recombination techniques take the form of plasmids. In the present description, "plasmid" and "vector" can be used exchangeably since the plasmid is the form of vector which is most frequently used. However, the invention is also intended to cover other forms of expression vectors, such as viral vectors, which exert similar functions. Furthermore, the term "vector" is also intended to encompass other vectors with which the skilled worker is familiar, such as phages, viruses such as SV40, CMV, TMV, transposons, IS elements, phasmids, phagemids, cosmids, linear or circular DNA.

[0130] The recombinant expression vectors advantageously used in the process comprise the nucleic acids described below or the above-described gene construct in a form which is suitable for expressing the nucleic acids used in a host cell, which means that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells used for the expression, which regulatory sequence(s) is/are linked operably with the nucleic acid sequence to be expressed. In a recombinant expression vector, "linked operably" means that the nucleotide sequence of interest is bound to the regulatory sequence(s) in such a way that the expression of the nucleotide sequence is possible and they are bound to each other in such a way that both sequences carry out the predicted function which is ascribed to the sequence (for example in an in-vitro transcription/translation system, or in a host cell if the vector is introduced into the host cell). The term "regulatory sequence" is intended to comprise promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), or see: Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., Ed.: Glick and Thompson, Chapter 7, 89-108, including the references cited therein. Regulatory sequences comprise those which govern the constitutive expression of a nucleotide sequence in many types of host cell and those which govern the direct expression of the nucleotide sequence only in specific host cells under specific conditions. The skilled worker knows that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the desired expression level of the protein and the like.

[0131] The recombinant expression vectors used can be designed for the expression of acyl-CoA:lysophospholipid acyltransferases, desaturases and elongases in prokaryotic or eukaryotic cells. This is advantageous since intermediate steps of the vector construction are frequently carried out in microorganisms for the sake of simplicity. For example, acyl-CoA:lysophospholipid acyltransferase, desaturase and elongase genes can be expressed in bacterial cells, insect cells (using Baculovirus expression vectors), yeast and other fungal cells (see Romanos, M. A., et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8:423-488; van den Hondel, C. A. M. J. J., et al. (1991) "Heterologous gene expression in filamentous fungi", in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology. 1, 3:239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Desaturaseudocohnilembus, Euplotes, Engelmaniella and Stylonychia, in particular of the genus Stylonychia lemnae, using vectors in a transformation method as described in WO 98/01572 and, preferably, in cells of multi-celled plants (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla., Chapter 6/7, pp. 71-119 (1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-43; Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (and references cited therein)). Suitable host cells are furthermore discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). As an alternative, the recombinant expression vector can be transcribed and translated in vitro, for example using T7-promoter regulatory sequences and T7-polymerase.

[0132] In most cases, the expression of proteins in prokaryotes involves the use of vectors comprising constitutive or inducible promoters which govern the expression of fusion or nonfusion proteins. Typical fusion expression vectors are, inter alia, pGEX (Pharmacia Biotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.), where glutathione S-transferase (GST), maltose-E binding protein and protein A, respectively, is fused with the recombinant target protein.

[0133] Examples of suitable inducible nonfusion E. coli expression vectors are, inter alia, pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). The target gene expression from the pTrc vector is based on the transcription from a hybrid trp-lac fusion promoter by the host RNA polymerase. The target gene expression from the vector pET 11d is based on the transcription of a T7-gn10-lac fusion promoter, which is mediated by a viral RNA polymerase (T7 gn1), which is coexpressed. This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident λ-prophage which harbors a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0134] Other vectors which are suitable for prokaryotic organisms are known to the skilled worker, these vectors are, for example in E. coli pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11 or pBdCl, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667.

[0135] In a further embodiment, the expression vector is a yeast expression vector. Examples for vectors for expression in the yeast S. cerevisiae comprise pYeDesaturasec1 (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and processes for the construction of vectors which are suitable for use in other fungi, such as the filamentous fungi, comprise those which are described in detail in: van den Hondel, C. A. M. J. J., & Punt, P. J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J. F. Peberdy et al., Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego]. Further suitable yeast vectors are, for example, pAG-1, YEp6, YEp13 or pEMBLYe23.

[0136] As an alternative, acyl-CoA:lysophospholipid acyltransferases, desaturases and/or elongases can be expressed in insect cells using Baculovirus expression vectors. Baculovirus vectors which are available for the expression of proteins in cultured insect cells (for example Sf9 cells) comprise the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0137] The abovementioned vectors offer only a small overview over suitable vectors which are possible. Further plasmids are known to the skilled worker and are described, for example, in: Cloning Vectors (Ed. Pouwels, P. H., et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). For further suitable expression systems for prokaryotic and eukaryotic cells, see the Chapters 16 and 17 in Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0138] In a further embodiment of the process, the acyl-CoA:lysophospholipid acyltransferases, desaturases and elongases can be expressed in single-celled plant cells (such as algae), see Falciatore et al., 1999, Marine Biotechnology 1 (3):239-251 and references cited therein, and in plant cells from higher plants (for example spermatophytes such as arable crops). Examples of plant expression vectors comprise those which are described in detail in: Becker, D., Kemper, E., Schell, J., and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20:1195-1197; and Bevan, M. W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, p. 15-38.

[0139] A plant expression cassette preferably comprises regulatory sequences which are capable of governing the expression of genes in plant cells and which are linked operably so that each sequence can fulfill its function, such as transcriptional termination, for example polyadenylation signals. Preferred polyadenylation signals are those which are derived from Agrobacterium tumefaciens T-DNA, such as gene 3 of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. 3 (1984) 835 et seq.), which is known as octopine synthase, or functional equivalents thereof, but all other terminator sequences which are functionally active in plants are also suitable.

[0140] Since plant gene expression is very often not limited to the transcriptional level, a plant expression cassette preferably comprises other sequences which are linked operably, such as translation enhancers, for example the overdrive sequence, which enhances the tobacco mosaic virus 5'-untranslated leader sequence, which increases the protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).

[0141] As described above, plant gene expression must be linked operably with a suitable promoter which triggers gene expression with the correct timing or in a cell- or tissue-specific manner. Utilizable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those which are derived from plant viruses, such as 35S CaMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), or plant promoters, such as the promoter of the small rubisco subunit, which is described in U.S. Pat. No. 4,962,028.

[0142] Other preferred sequences for use in operable linkage in plant gene expression cassettes are targeting sequences, which are required for steering the gene product into its corresponding cell compartment (see a review in Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited therein), for example into the vacuole, into the nucleus, all types of plastids, such as amyloplasts, chloroplasts, chromoplasts, the extracellular space, the mitochondria, the endoplasmid reticulum, elaioplasts, peroxisomes and other compartments of plant cells.

[0143] As described above, plant gene expression can also be achieved via a chemically inducible promoter (see review in Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired that the gene expression takes place in a time-specific manner. Examples of such promoters are a salicylic-acid-inducible promoter (WO 95/19443), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404) and an ethanol-inducible promoter.

[0144] Promoters which respond to biotic or abiotic stress conditions are also suitable, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the heat-inducible tomato hsp80 promoter (U.S. Pat. No. 5,187,267), the chill-inducible potato alpha-amylase promoter (WO 96/12814) or the wound-inducible pinII promoter (EP-A-0 375 091).

[0145] Especially preferred are those promoters which bring about the gene expression in tissues and organs in which the biosynthesis of fatty acids, lipids and oils takes place, in seed cells, such as cells of the endosperm and of the developing embryo. Suitable promoters are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980) or the legumine B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and promoters which bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Suitable noteworthy promoters are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230) or the promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamine gene, the wheat gliadine gene, the wheat glutelin gene, the maize zeine gene, the oat glutelin gene, the sorghum kasirin gene or the rye secalin gene, which are described in WO 99/16890.

[0146] In particular, it may be desired to bring about the multiparallel expression of the acyl-CoA:lysophospholipid acyltransferases used in the process alone or in combination with desaturases and/or elongases. Such expression cassettes can be introduced via the simultaneous transformation of a plurality of individual expression constructs or, preferably, by combining a plurality of expression cassettes on one construct. Also, a plurality of vectors can be transformed with in each case a plurality of expression cassettes and then transferred into the host cell.

[0147] Other promoters which are likewise especially suitable are those which bring about plastid-specific expression, since plastids constitute the compartment in which the precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters, such as the viral RNA polymerase promoter, are described in WO 95/16783 and WO 97/06250, and the clpP promoter from Arabidopsis, described in WO 99/46394.

[0148] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection", conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of methods known in the prior art for the introduction of foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and other laboratory textbooks such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.

[0149] Host cells which are suitable in principle for taking up the nucleic acid according to the invention, the gene product according to the invention or the vector according to the invention are all prokaryotic or eukaryotic organisms. The host organisms which are advantageously used are microorganisms such as fungi or yeasts, or plant cells, preferably plants or parts thereof. Fungi, yeasts or plants are preferably used, especially preferably plants, very especially preferably plants such as oil crops, which are high in lipid compounds, such as oilseed rape, evening primrose, hemp, thistle, peanut, canola, linseed, soybean, safflower, sunflower, borage, or plants such as maize, wheat, rye, oats, triticale, rice, barley, cotton, cassava, pepper, Tagetes, Solanacea plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), and perennial grasses and fodder crops. Especially preferred plants according to the invention are oil crops such as soybean, peanut, oilseed rape, canola, linseed, hemp, evening primrose, sunflower, safflower, trees (oil palm, coconut).

[0150] The invention furthermore relates to isolated nucleic acid sequences as described above coding for polypeptides having acyl-CoA:lysophospholipid-acyltransferase activity, wherein the acyl-CoA:lysophospholipid acyltransferases encoded by said nucleic acid sequences specifically convert C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids having at least one double bond in the fatty acid molecule.

[0151] Advantageous isolated nucleic acid sequences are sequences selected from the group consisting of:

[0152] a) a nucleic acid sequence having the sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7,

[0153] b) nucleic acid sequences which can be derived from the coding sequence comprised in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 as a result of the degenerated genetic code

[0154] c) derivatives of the nucleic acid sequence depicted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 which code for polypeptides having the amino acid sequence depicted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and are at least 40% homologous at the amino acid level to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 and have an acyl-CoA:lysophospholipid-acyltransferase activity.

[0155] The abovementioned nucleic acids according to the invention are derived from organisms such as animals, ciliates, fungi, plants such as algae or dinoflagellates which are capable of synthesizing PUFAs.

[0156] In an advantageous embodiment, the term "nucleic acid (molecule)" as used in the present context additionally comprises the untranslated sequence at the 3' and at the 5' end of the coding gene region: at least 500, preferably 200, especially preferably 100 nucleotides of the sequence upstream of the 5' end of the coding region and at least 100, preferably 50, especially preferably 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. An "isolated" nucleic acid molecule is separate from other nucleic acid molecules which are present in the natural source of the nucleic acid. An "isolated" nucleic acid preferably has no sequences which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (for example sequences which are located at the 5' and 3' ends of the nucleic acid). In various embodiments, the isolated acyl-CoA:lysophospholipid acyltransferase molecule can comprise for example fewer than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0157] The nucleic acid molecules used in the process, for example a nucleic acid molecule with a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or of a part thereof can be isolated using molecular-biological standard techniques and the sequence information provided herein. Also, for example a homologous sequence or homologous, conserved sequence regions can be identified at the DNA or amino acid level with the aid of comparative algorithms. They can be used as hybridization probe together with standard hybridization techniques (such as, for example, those described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for isolating further nucleic acid sequences which can be used in the process. Moreover, a nucleic acid molecule comprising a complete sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or a part thereof can be isolated by polymerase chain reaction, where oligonucleotide primers which are based on this sequence or on parts thereof are used (for example a nucleic acid molecule comprising the complete sequence or part thereof can be isolated by polymerase chain reaction using oligonucleotide primers which have been generated based on this same sequence). For example, mRNA can be isolated from cells (for example by means of the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA by means of reverse transcriptase (for example Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for the amplification by means of polymerase chain reaction can be generated based on one of the sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or with the aid of the amino acid sequences detailed in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A nucleic acid according to the invention can be amplified by standard PCR amplification techniques using cDNA or, alternatively, genomic DNA as template and suitable oligonucleotide primers. The nucleic acid amplified thus can be cloned into a suitable vector and characterized by means of DNA sequence analysis. Oligonucleotides which correspond to a desaturase nucleotide sequence can be generated by standard synthetic methods, for example using an automatic DNA synthesizer.

[0158] Homologs of the acyl-CoA:lysophospholipid acyltransferase nucleic acid sequences with the sequence SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 means, for example, allelic variants with at least approximately 40 to 60%, preferably at least approximately from 60 to 70%, more preferably at least approximately from 70 to 80%, 80% to 90% or 90 to 95% and even more preferably at least approximately 95%, 96%, 97%, 98%, 99% or more homology with a nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or its homologs, derivatives or analogs or parts thereof. Furthermore, isolated nucleic acid molecules of a nucleotide sequence which hybridize with one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or with a part thereof, for example hybridized under stringent conditions. Allelic variants comprise in particular functional variants which can be obtained by deletion, insertion or substitution of nucleotides from/into the sequence detailed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, it being intended, however, that the enzyme activity of the resulting proteins which are synthesized is advantageously retained for the insertion of one or more genes. Proteins which retain the enzymatic activity of acyl-CoA:lysophospholipid acyltransferase, i.e. whose activity is essentially not reduced, means proteins with at least 10%, preferably 20%, especially preferably 30%, very especially preferably 40% of the original enzyme activity in comparison with the protein encoded by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0159] Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 means for example also bacterial, fungal and plant homologs, truncated sequences, single-stranded DNA or RNA of the coding and noncoding DNA sequence.

[0160] Homologs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 also mean derivatives such as, for example, promoter variants. The promoters upstream of the nucleotide sequences detailed can be modified by one or more nucleotide exchanges, by insertion(s) and/or deletion(s) without the functionality or activity of the promoters being adversely affected, however. It is furthermore possible that the modification of the promoter sequence enhances their activity or that they are replaced entirely by more active promoters, including those from heterologous organisms.

[0161] The abovementioned nucleic acids and protein molecules with acyl-CoA:lysophospholipid acyltransferase activity which are involved in the metabolism of lipids and fatty acids, PUFA cofactors and enzymes or in the transport of lipophilic compounds across membranes are used in the process according to the invention for the modulation of the production of PUFAs in transgenic organisms, advantageously in plants, such as maize, wheat, rye, oats, triticale, rice, barley, soybean, peanut, cotton, Linum species such as linseed or flax, Brassica species such as oilseed rape, canola and turnip rape, pepper, sunflower, borage, evening primrose and Tagetes, Solanaceae plants such as potato, tobacco, eggplant and tomato, Vicia species, pea, cassava, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut) and perennial grasses and fodder crops, either directly (for example when the overexpression or optimization of a fatty acid biosynthesis protein has a direct effect on the yield, production and/or production efficiency of the fatty acid from modified organisms) and/or can have an indirect effect which nevertheless leads to an enhanced yield, production and/or production efficiency of the PUFAs or a reduction of undesired compounds (for example when the modulation of the metabolism of lipids and fatty acids, cofactors and enzymes leads to modifications of the yield, production and/or production efficiency or the composition of the desired compounds within the cells, which, in turn, can affect the production of one or more fatty acids).

[0162] The combination of various precursor molecules and biosynthesis enzymes leads to the production of various fatty acid molecules, which has a decisive effect on lipid composition, since polyunsaturated fatty acids (═PUFAs) are not only incorporated into triacylglycerol but also into membrane lipids.

[0163] Lipid synthesis can be divided into two sections: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate, and the addition or modification of a polar head group. Usual lipids which are used in membranes comprise phospholipids, glycolipids, sphingolipids and phosphoglycerides. Fatty acid synthesis starts with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA carboxylase or into acetyl-ACP by acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted via a series of condensation, reduction and dehydratization reactions so that a saturated fatty acid molecule with the desired chain length is obtained. The production of the unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically by means of molecular oxygen or anaerobically (regarding the fatty acid synthesis in microorganisms, see F. C. Neidhardt et al. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., pp. 612-636 and references cited therein; Lengeler et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York, and the references therein, and Magnuson, K., et al. (1993) Microbiological Reviews 57:522-542 and the references therein). To undergo the further elongation steps, the resulting phospholipid-bound fatty acids must be returned to the fatty acid CoA ester pool. This is made possible by acyl-CoA:lysophospholipid acyltransferases. Moreover, these enzymes are capable of transferring the elongated fatty acids from the CoA esters back to the phospholipids. If appropriate, this reaction sequence can be followed repeatedly (see FIG. 10).

[0164] Examples of precursors for the biosynthesis of PUFAs are oleic acid, linoleic acid and linolenic acid. These C₁₈-carbon fatty acids must be elongated to C₂₀ and C₂₂ in order to obtain fatty acids of the eicosa and docosa chain type. With the aid of the acyl-CoA:lysophospholipid acyltransferases used in the process, advantageous in combination with desaturases such as Δ4-, Δ5-, Δ6- and Δ8-desaturases and/or Δ5-, Δ6-, Δ9-elongases, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid and various other long-chain PUFAs can be obtained, extracted and employed in various applications regarding foodstuffs, feedstuffs, cosmetics or pharmaceuticals. Preferably C₁₈-, C₂₀- and/or C₂₂-fatty acids with at least two, advantageously at least three, four, five or six, double bonds in the fatty acid molecule can be prepared using the abovementioned enzymes, to give preferably C₂₀- or C₂₂-fatty acids with advantageously three, four or five double bonds in the fatty acid molecule. Desaturation may take place before or after elongation of the fatty acid in question. This is why the products of the desaturase activities and the further desaturation and elongation steps which are possible result in preferred PUFAs with a higher degree of desaturation, including a further elongation from C₂₀- to C₂₂-fatty acids, to fatty acids such as γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, stearidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. Substrates of the acyl-CoA:lysophospholipid acyltransferases used in the process according to the invention are C₁₆-, C₁₈-, C₂₀- or C₂₂-fatty acids such as, for example, palmitic acid, palmitoleic acid, linoleic acid, γ-linolenic acid, α-linolenic acid, dihomo-γ-linolenic acid, eicosatetraenoic acid or stearidonic acid. Preferred substrates are linoleic acid, γ-linolenic acid and/or α-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosatetraenoic acid or eicosapentaenoic acid. The C₁₈-, C₂₀- or C₂₂-fatty acids with at least two double bonds in the fatty acid are obtained in the process according to the invention in the form of the free fatty acid or in the form of their esters, for example in the form of their glycerides.

[0165] The term "glyceride" is understood as meaning glycerol esterified with one, two or three carboxyl radicals (mono-, di- or triglyceride). "Glyceride" is also understood as meaning a mixture of various glycerides. The glyceride or glyceride mixture may comprise further additions, for example free fatty acids, antioxidants, proteins, carbohydrates, vitamins and/or other substances.

[0166] For the purposes of the invention, a "glyceride" is furthermore understood as meaning glycerol derivatives. In addition to the above-described fatty acid glycerides, these also include glycerophospholipids and glyceroglycolipids. Preferred examples which may be mentioned in this context are the glycerophospholipids such as lecithin (phosphatidylcholine), cardiolipin, phosphatidylglycerol, phosphatidylserine and alkylacylglycerophospholipids.

[0167] Furthermore, fatty acids must subsequently be translocated to various modification sites and incorporated into the triacylglycerol storage lipid. A further important step in lipid synthesis is the transfer of fatty acids to the polar head groups, for example by glycerol fatty acid acyltransferase (see Frentzen, 1998, Lipid, 100(4-5):161-166).

[0168] Publications on plant fatty acid biosynthesis and on the desaturation, the lipid metabolism and the membrane transport of lipidic compounds, on beta-oxidation, fatty acid modification and cofactors, triacylglycerol storage and triacylglycerol assembly, including the references therein, see the following papers: Kinney, 1997, Genetic Engineering, Ed.: J K Setlow, 19:149-166; Ohlrogge and Browse, 1995, Plant Cell 7:957-970; Shanklin and Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641; Voelker, 1996, Genetic Engineering, Ed.: J K Setlow, 18:111-13; Gerhardt, 1992, Prog. Lipid R. 31:397-417; Guhnemann-Schafer & Kindl, 1995, Biochim. Biophys Acta 1256:181-186; Kunau et al., 1995, Prog. Lipid Res. 34:267-342; Stymne et al., 1993, in: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, Ed.: Murata and Somerville, Rockville, American Society of Plant Physiologists, 150-158, Murphy & Ross 1998, Plant Journal. 13(1):1-16.

[0169] The PUFAs produced in the process comprise a group of molecules which higher animals are no longer capable of synthesizing and must therefore take up, or which higher animals are no longer capable of synthesizing themselves in sufficient quantity and must therefore take up additional quantities, although they can be synthesized readily by other organisms such as bacteria; for example, cats are no longer capable of synthesizing arachidonic acid.

[0170] The term "acyl-CoA:lysophospholipid acyltransferases" comprises for the purposes of the invention proteins which participate in the transfer of the fatty acids bound to phospholipids to the CoA-ester pool and vice versa and their homologs, derivatives and analogs. Phospholipids for the purposes of the invention are understood as meaning phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and/or phosphatidylinositol, advantageously phosphatidylcholine. The terms acyl-CoA:lysophospholipid acyltransferase(s) comprise nucleic acid sequences which encode an acyl-CoA:lysophospholipid acyltransferase and part of which may be a coding region and likewise corresponding 5' and 3' untranslated sequence regions. The terms production or productivity are known in the art and encompass the concentration of the fermentation product (compounds of the formula I) which is formed within a specific period of time and in a specific fermentation volume (for example kg of product per hour per liter). The term production efficiency comprises the time required for obtaining a specific production quantity (for example the time required by the cell to establish a certain throughput rate of a fine chemical). The term yield or product/carbon yield is known in the art and comprises the efficiency of the conversion of the carbon source into the product (i.e. the fine chemical). This is usually expressed for example as kg of product per kg of carbon source. By increasing the yield or production of the compound, the amount of the molecules obtained of this compound, or of the suitable molecules of this compound obtained in a specific culture quantity over a specified period of time is increased. The terms biosynthesis or biosynthetic pathway are known in the art and comprise the synthesis of a compound, preferably an organic compound, by a cell from intermediates, for example in a multi-step and strongly regulated process. The terms catabolism or catabolic pathway are known in the art and comprise the cleavage of a compound, preferably of an organic compound, by a cell to give catabolites (in more general terms, smaller or less complex molecules), for example in a multi-step and strongly regulated process. The term metabolism is known in the art and comprises the totality of the biochemical reactions which take place in an organism. The metabolism of a certain compound (for example the metabolism of a fatty acid) thus comprises the totality of the biosynthetic pathways, modification pathways and catabolic pathways of this compound in the cell which relate to this compound.

[0171] In a further embodiment, derivatives of the nucleic acid molecule according to the invention represented in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 encode proteins with at least 40%, advantageously from approximately 50 to 60%, advantageously at least from approximately 60 to 70% and more preferably at least from approximately 70 to 80%, 80 to 90%, 90 to 95% and most preferably at least approximately 96%, 97%, 98%, 99% or more homology (=identity) with a complete amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. The homology was calculated over the entire amino acid or nucleic acid sequence region. The program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981)], which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991)], were used for the sequence alignment. The sequence homology values which are indicated above as a percentage were determined over the entire sequence region using the program BestFit and the following settings: Gap Weight: 8, Length Weight: 2.

[0172] Moreover, the invention comprises nucleic acid molecules which differ from one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 (and parts thereof) owing to the degeneracy of the genetic code and which thus encode the same acyl-CoA:lysophospholipid acyltransferase as those encoded by the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0173] In addition to the acyl-CoA:lysophospholipid acyltransferase(s) shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, the skilled worker will recognize that DNA sequence polymorphisms which lead to changes in the amino acid sequences of the acyl-CoA:lysophospholipid acyltransferase(s) may exist within a population. These genetic polymorphisms in the acyl-CoA:lysophospholipid acyltransferase gene may exist between individuals within a population owing to natural variation. These natural variants usually bring about a variance of 1 to 5% in the nucleotide sequence of the acyl-CoA:lysophospholipid acyltransferase gene. Each and every one of these nucleotide variations and resulting amino acid polymorphisms in the acyl-CoA:lysophospholipid acyltransferase which are the result of natural variation and do not modify the functional activity of acyl-CoA:lysophospholipid acyltransferases are to be encompassed by the invention.

[0174] Owing to their homology to the acyl-CoA:lysophospholipid acyltransferase nucleic acids disclosed here, nucleic acid molecules which are advantageous for the process according to the invention can be isolated following standard hybridization techniques under stringent hybridization conditions, using the sequences or part thereof as hybridization probe. In this context it is possible, for example, to use isolated nucleic acid molecules which are least 15 nucleotides in length and which hybridize under stringent conditions with the nucleic acid molecules which comprise a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. Nucleic acids with at least 25, 50, 100, 250 or more nucleotides can also be used. The term "hybridizes under stringent conditions" as used in the present context is intended to describe hybridization and washing conditions under which nucleotide sequences with at least 60% homology to one another usually remain hybridized with one another. Conditions are preferably such that sequences with at least approximately 65%, preferably at least approximately 70% and especially preferably at least 75% or more homology to one another usually remain hybridized to one another. These stringent conditions are known to the skilled worker and described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred nonlimiting example of stringent hybridization conditions is hybridizations in 6× sodium chloride/sodium citrate (=SSC) at approximately 45° C., followed by one or more washing steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, regarding temperature and buffer concentration. Under "standard hybridization conditions", for example, the hybridization temperature is, depending on the type of nucleic acid, between 42° C. and 58° C. in aqueous buffer with a concentration of 0.1 to 5×SSC (pH 7.2). If organic solvent, for example 50% formamide, is present in the abovementioned buffer, the temperature under standard conditions is approximately 42° C. The hybridization conditions for DNA:DNA hybrids, for example, are 0.1×SSC and 20° C. to 45° C., preferably 30° C. to 45° C. The hybridization conditions for DNA:RNA hybrids are, for example, 0.1×SSC and 30° C. to 55° C., preferably 45° C. to 55° C. The abovementioned hybridization temperatures are determined by way of example for a nucleic acid with approximately 100 bp (=base pairs) in length and with a G+C content of 50% in the absence of formamide. The skilled worker knows how to determine the required hybridization conditions on the basis of the abovementioned textbooks or textbooks such as Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford.

[0175] In order to determine the percentage of homology (=identity) of two amino acid sequences (for example one of the sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8) or of two nucleic acids (for example SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7), the sequences are written one under the other for an optimal comparison (for example, gaps may be introduced into the sequence of a protein or of a nucleic acid in order to generate an optimal alignment with the other protein or the other nucleic acid). Then, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in a sequence is occupied by the same amino acid residue or the same nucleotide as the corresponding position in the other sequence, then the molecules are homologous at this position (i.e. amino acid or nucleic acid "homology" as used in the present context corresponds to amino acid or nucleic acid "identity"). The percentage of homology between the two sequences is a function of the number of positions which the sequences share (i.e. % homology=number of identical positions/total number of positions×100). The terms homology and identity are therefore to be considered as synonymous.

[0176] An isolated nucleic acid molecule which encodes an acyl-CoA:lysophospholipid acyltransferase which is homologous to a protein sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 can be generated by introducing one or more nucleotide substitutions, additions or deletions in/into a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 so that one or more amino acid substitutions, additions or deletions are introduced in/into the protein which is encoded. Mutations in one of the sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 can be introduced by standard techniques such as site-specific mutagenesis and PCR-mediated mutagenesis. It is preferred to generate conservative amino acid substitutions in one or more of the predicted nonessential amino acid residues. In a "conservative amino acid substitution", the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families comprise amino acids with basic side chains (for example lysine, arginine, histidine), acidic side chains (for example aspartic acid, glutamic acid), uncharged polar side chains (for example glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), unpolar side chains (for example alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (for example threonine, valine, isoleucine) and aromatic side chains (for example tyrosine, phenylalanine, tryptophan, histidine). A predicted nonessential amino acid residue in an acyl-CoA:lysophospholipid acyltransferase is thus preferably replaced by another amino acid residue from the same family of side chains. In another embodiment, the mutations can, alternatively, be introduced randomly over all or part of the sequence encoding the acyl-CoA:lysophospholipid acyltransferase, for example by saturation mutagenesis, and the resulting mutants can be screened by the herein-described acyl-CoA:lysophospholipid acyltransferase activity in order to identify mutants which have retained the acyl-CoA:lysophospholipid acyltransferase activity. Following the mutagenesis of one of the sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, the protein which is encoded can be expressed recombinantly, and the activity of the protein can be determined, for example using the tests described in the present text.

[0177] The present invention is illustrated in greater detail by the examples which follow, which are not to be construed as limiting. The content of all of the references, patent applications, patents and published patent applications cited in the present patent application is herewith incorporated by reference.

EXAMPLES

Example 1

General Methods

a) General Cloning Methods:

[0178] Cloning methods such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of Escherichia coli and yeast cells, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994) "Methods in Yeast Genetics" (Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3).

b) Chemicals

[0179] Unless stated otherwise in the text, the chemicals used were obtained in analytical-grade quality from Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Serva (Heidelberg, Germany) and Sigma (Deisenhofen, Germany). Solutions were prepared using purified, pyrogen-free water, referred to as H₂O hereinbelow, from a Milli-Q Water System water purification system (Millipore, Eschborn, Germany). Restriction endonucleases, DNA-modifying enzymes and molecular-biological kits were obtained from AGS (Heidelberg, Germany), Amersham (Brunswick, Germany), Biometra (Gottingen, Germany), Boehringer (Mannheim, Germany), Genomed (Bad Oeynhausen, Germany), New England Biolabs (Schwalbach/Taunus, Germany), Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt, Germany), Pharmacia (Freiburg, Germany), Qiagen (Hilden, Germany) and Stratagene (Amsterdam, the Netherlands). Unless stated otherwise, they were used according to the manufacturer's instructions.

c) Cloning and Expression of Desaturases and Elongases

[0180] The Escherichia coli strain XL1 Blue MRF' kan (Stratagene) was used for subcloning Δ6-desaturase from Physcomitrella patens. This gene was functionally expressed using the Saccharomyces cerevisiae strain INVSc 1 (Invitrogen Co.). E. coli was cultured in Luria-Bertani broth (LB, Duchefa, Haarlem, the Netherlands) at 37° C. If necessary, ampicillin (100 mg/liter) was added and 1.5% (w/v) agar was added for solid LB media. S. cerevisiae was cultured at 30° C. either in YPG medium or in complete minimal medium without uracil (CMdum; see in: Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Albright, L. B., Coen, D. M., and Varki, A. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York) with either 2% (w/v) raffinose or glucose. For solid media, 2% (w/v) Bacto®-Agar (Difco) were added. The plasmids used for cloning and expression are pUC18 (Pharmacia) and pYES2 (Invitrogen Co.).

d) Cloning and Expression of PUFA-Specific Desaturases and Elongases

[0181] For expression in plants, cDNA clones of SEQ ID NO: 9, 11 or 13 were modified so as for only the coding region to be amplified by means of polymerase chain reaction with the aid of two oligonucleotides. Care was taken here to observe a consensus sequence upstream of the start codon, for efficient translation. To this end, either the ATA or the AAA base sequence was chosen and inserted into the sequence upstream of the ATG [Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-2929]. In addition, a restriction cleavage site was introduced upstream of this consensus triplet, which must be compatible with the cleavage site of the target vector into which the fragment is to be cloned and with the aid of which gene expression is to be carried out in microorganisms or plants.

[0182] The PCR reaction was carried out in a thermocycler (Biometra), using plasmid DNA as template and Pfu DNA polymerase (Stratagene) and the following temperature program: 3 min at 96° C., followed by 30 cycles of 30 s at 96° C., 30 s at 55° C. and 2 min at 72° C., 1 cycle of 10 min at 72° C. and stop at 4° C. The annealing temperature was varied depending on the oligonucleotides chosen. A synthesis time of about one minute per kilobase pair of DNA has to be taken as starting point. Other parameters which influence the PCR, such as, for example, Mg ions, salt, DNA polymerase etc., are familiar to the skilled worker in the field and may be varied as required.

[0183] The correct size of the amplified DNA fragment was confirmed by means of agarose-TBE gel electrophoresis. The amplified DNA was extracted from the gel using the QIAquick gel extraction kit (QIAGEN) and ligated into the SmaI restriction site of the dephosphorylated pUC18 vector, using the Sure Clone Ligations Kit (Pharmacia), resulting in the pUC derivatives. After transformation of E. coli XL1 Blue MRF' kan a DNA minipreparation [Riggs, M. G., & McLachlan, A. (1986) A simplified screening procedure for large numbers of plasmid mini-preparation. BioTechniques 4, 310-313] of ampicillin-resistant transformants was carried out, and positive clones were identified by means of BamHI restriction analysis. The sequence of the cloned PCR product was confirmed by means of resequencing using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).

e) Transformation of Agrobacterium

[0184] Unless described otherwise, Agrobacterium-mediated plant transformation was carried out with the aid of an Agrobacterium tumefaciens strain, as by Deblaere et al. (1984, Nucl. Acids Res. 13, 4777-4788).

f) Plant Transformation

[0185] Unless described otherwise, Agrobacterium-mediated plant transformation was carried out using standard transformation and regeneration techniques (Gelvin, Stanton B., Schilperoort, Robert A., Plant Molecular Biology Manual, 2nd ed., Dordrecht: Kluwer Academic Publ., 1995, in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R., Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993, 360 S., ISBN 0-8493-5164-2).

[0186] According thereto, it is possible to transform, for example, oilseed rape by means of cotyledon or hypocotyl transformation (Moloney et al., Plant Cell 8 (1989) 238-242; De Block et al., Plant Physiol. 91 (1989) 694-701). The use of antibiotics for the selection of agrobacteria and plants depends on the binary vector used for transformation and the Agrobacterium strain. Normally, oilseed rape is selected using kanamycin as selectable plant marker.

[0187] The transformation of soya may be carried out using, for example, a technique described in EP-A-0 0424 047 (Pioneer Hi-Bred International) or in EP-A-0 0397 687, U.S. Pat. No. 5,376,543, U.S. Pat. No. 5,169,770 (University Toledo).

[0188] The transformation of plants using particle bombardment, polyethylene glycol-mediated DNA uptake or via the silicon carbonate fiber technique is described, for example, by Freeling and Walbot "The maize handbook" (1993) ISBN 3-540-97826-7, Springer Verlag New York).

[0189] Unless described otherwise, Agrobacterium-mediated gene transfer into linseed (Linum usitatissimum) was carried out by the technique as described in Mlynarova et al. [(1994) Plant Cell Report 13:282-285].

g) Plasmids for Plant Transformation

[0190] Binary vectors based on the vectors pBinAR (Hofgen and Willmitzer, Plant Science 66 (1990) 221-230) or pGPTV (Becker et al. 1992, Plant Mol. Biol. 20:1195-1197) were used for plant transformation. The binary vectors which comprise the nucleic acids to be expressed are constructed by ligating the cDNA in sense orientation into the T-DNA. 5' of the cDNA, a plant promoter activates cDNA transcription. A polyadenylation sequence is located 3' of the cDNA. The binary vectors may carry different marker genes such as, for example, the acetolactate synthase gene (AHAS or ALS) [Ott et al., J. Mol. Biol. 1996, 263:359-360] which imparts a resistance to the imidazolinones or the nptII marker gene which codes for a kanamycin resistance imparted by neomycin phosphotransferase.

[0191] Tissue-specific expression of the nucleic acids can be achieved using a tissue-specific promoter. Unless described otherwise, the LeB4 or the USP promoter or the phaseolin promoter was cloned 5' of the cDNA. Terminators used were the NOS terminator and the OCS terminator (see FIG. 8). FIG. 8 depicts a vector map of the vector used for expression, pSUN3CeLPLAT.

[0192] It is also possible to use any other seed-specific promoter element such as, for example, the napin or arcelin promoter (Goossens et al. 1999, Plant Phys. 120(4):1095-1103 and Gerhardt et al. 2000, Biochimica et Biophysica Acta 1490(1-2):87-98).

[0193] The CaMV-355 promoter or a v-ATPase C1 promoter can be used for constitutive expression in the whole plant.

[0194] The nucleic acids used in the process which encode acyl-CoA:lysophospholipid acyltransferases; desaturases or elongases were cloned into a binary vector one after the other by constructing a plurality of expression cassettes, in order to mimic the metabolic pathway in plants.

[0195] Within an expression cassette, the protein to be expressed may be guided into a cellular compartment by using a signal peptide, for example for plastids, mitochondria or the endoplasmic reticulum (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996) 285-423). The signal peptide is cloned 5' of and in frame with the cDNA in order to achieve the subcellular localization of the fusion protein.

[0196] Examples of multiexpression cassettes were disclosed in DE 102 19 203 and are given again below.

i.) Promoter-Terminator Cassettes

[0197] Expression cassettes consist of at least two functional units such as a promoter and a terminator. Further desired gene sequences such as targeting sequences, coding regions of genes or parts thereof etc. may be inserted between promoter and terminator. To construct the expression cassettes, promoters and terminators (USP promoter: Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67); OCS terminator: Gielen et al. EMBO J. 3 (1984) 835ff.) were isolated with the aid of the polymerase chain reaction and tailor-made with flanking sequences of choice on the basis of synthetic oligonucleotides.

[0198] Examples of oligonucleotides which may be used are the following:

TABLE-US-00001 USP1 upstream (SEQ ID NO: 38): CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA- USP2 upstream (SEQ ID NO: 39): CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA- USP3 upstream (SEQ ID NO: 40): CCGGAATTCGGCGCGCCGAGCTCCTCGAGCAAATTTACACATTGCCA- USP1 downstream (SEQ ID NO: 41): AAAACTGCAGGCGGCCGCCCACCGCGGTGGGCTGGCTATGAAGAAATT- USP2 downstream (SEQ ID NO: 42): CGCGGATCCGCTGGCTATGAAGAAATT- USP3 downstream (SEQ ID NO: 43): TCCCCCGGGATCGATGCCGGCAGATCTGCTGGCTATGAAGAAATT- OCS1 upstream (SEQ ID NO: 44): AAAACTGCAGTCTAGAAGGCCTCCTGCTTTAATGAGATAT- OCS2 upstream (SEQ ID NO: 45): CGCGGATCCGATATCGGGCCCGCTAGCGTTAACCCTGCTTTAATGAGATAT- OCS3 upstream (SEQ ID NO: 46): TCCCCCGGGCCATGGCCTGCTTTAATGAGATAT- OCS1 downstream (SEQ ID NO: 47): CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA- OCS2 downstream (SEQ ID NO: 48): CCCAAGCTTGGCGCGCCGAGCTCGAATTCGTCGACGGACAATCAGTAAATTGA- OCS3 downstream (SEQ ID NO: 49): CCCAAGCTTGGCGCGCCGAGCTCGTCGACGGACAATCAGTAAATTGA-

[0199] The methods are known to the skilled worker in the field and are well known from the literature.

[0200] In a first step, a promoter and a terminator were amplified via PCR. The terminator was then cloned into a recipient plasmid and, in a second step, the promoter was inserted upstream of the terminator. As a result, an expression cassette was cloned into the basic plasmid. The plasmids pUT1, 2 and 3 were thus generated on the basis of the pUC19 plasmid.

[0201] The corresponding constructs or plasmids are defined in SEQ ID NO: 15, 16 to 17. They comprise the USP promoter and the OCS terminator. Based on these plasmids, the construct pUT12 was generated by cutting pUT1 by means of SaII/ScaI and pUT2 by means of XhoI/ScaI. The fragments comprising the expression cassettes were ligated and transformed into E. coli XL1 blue MRF. After isolating ampicillin-resistant colonies, DNA was prepared and those clones which comprise two expression cassettes were identified by restriction analysis. The XhoI/SaII ligation of compatible ends has eliminated here the two cleavage sites, XhoI and SaII, between the expression cassettes. The resulting plasmid, pUT12, is indicated in SEQ ID NO: 18. Subsequently, pUT12 was cut again by means of SaI/ScaI and pUT3 was cut by means of XhoI/ScaI. The fragments comprising the expression cassettes were ligated and transformed into E. coli XLI blue MRF. After isolation from ampicillin-resistant colonies, DNA was again prepared, and those clones which comprise three expression cassettes were identified by restriction analysis. In this manner, a set of multiexpression cassettes was produced which can be utilized for insertion of desired DNA and which is described in table 1 and which moreover can incorporate further expression cassettes.

[0202] Said cassettes comprise the following elements:

TABLE-US-00002 TABLE 1 Cleavage sites Cleavage sites PUC19 upstream of the USP Multiple downstream of the OCS derivative promoter cloning cleavage sites terminator PUT1 EcoRI/AscI/SacI/XhoI BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ HindIII PUT2 EcoRI/AscI/SacI/XhoI BamHI/EcoRV/ApaI/NheI/HpaI SalI/EcoRI/SacI/AscI/ HindIII PUT3 EcoRI/AscI/SacI/XhoI BglII/NaeI/ClaI/SmaI/NcoI SalI/SacI/AscI/HindIII PUT12 EcoRI/AscI/SacI/XhoI BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ double and HindIII expression BamHI/EcoRV/ApaI/NheI/HpaI cassette PUT123 EcoRI/AscI/SacI/XhoI 1. BstXI/NotI/PstI/XbaI/StuI SalI/SacI/AscI/HindIII triple and expression 2. BamHI/EcoRV/ApaI/NheI/HpaI cassette and 3. BglII/NaeI/ClaI/SmaI/NcoI

[0203] Furthermore, further multiexpression cassettes may be generated, as described and as specified in more detail in table 2, with the aid of the

[0204] i) USP promoter or with the aid of the

[0205] ii) 700 base pair 3' fragment of the LeB4 promoter or with the aid of the

[0206] iii) DC3 promoter and employed for seed-specific gene expression.

[0207] The DC3 promoter is described in Thomas, Plant Cell 1996, 263:359-368 and consists merely of the region from -117 to +26, which is why it therefore constitutes one of the smallest known seed-specific promoters. The expression cassettes may comprise several copies of the same promoter or else be constructed via three different promoters.

[0208] Advantageously used polylinker- or polylinker-terminator-polylinkers can be found in the sequences SEQ ID NO: 23 to 25.

TABLE-US-00003 TABLE 2 Multiple expression cassettes Plasmid name of Cleavage sites Cleavage sites the pUC19 upstream of the Multiple downstream of the derivative particular promoter cloning cleavage sites OCS terminator pUT1 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ (pUC19 with HindIII USP-OCS1) PDCT EcoRI/AscI/SacI/XhoI (2) BamHI/EcoRV/ApaI/NheI/ SalI/EcoRI/SacI/AscI/ (pUC19 with HpaI HindIII DC3-OCS) PleBT EcoRI/AscI/SacI/XhoI (3) BglII/NaeI/ClaI/SmaI/NcoI SalI/SacI/AscI/HindIII (pUC19 with LeB4(700)-OCS) PUD12 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI SalI/EcoRI/SacI/AscI/ (pUC 19 with and HindIII USP-OCS1 and (2) BamHI/EcoRV/ApaI/NheI/ with DC3-OCS) HpaI PUDL123 EcoRI/AscI/SacI/XhoI (1) BstXI/NotI/PstI/XbaI/StuI and SalI/SacI/AscI/HindIII Triple expression (2) BamHI/(EcoRV*)/ApaI/NheI/ cassette HpaI and (pUC19 with (3) BglII/NaeI/ClaI/SmaI/NcoI USP/DC3 and LeB4-700) *EcoRV cleavage site cuts in the 700 base pair fragment of the LeB4 promoter (LeB4-700)

[0209] Further promoters for multigene constructs can be generated analogously, in particular by using the

a) 2.7 kB fragment of the LeB4 promoter or with the aid of the b) phaseolin promoter or with the aid of the c) constitutive v-ATPase c1 promoter.

[0210] It may be particularly desirable to use further particularly suitable promoters for constructing seed-specific multiexpression cassettes, such as, for example, the napin promoter or the arcelin-5 promoter.

[0211] Further vectors which can be utilized in plants and which have one or two or three promoter-terminator expression cassettes can be found in the sequences SEQ ID NO: 26 to SEQ ID NO: 31.

ii.) Generation of Expression Constructs which Comprise Promoter, Terminator and Desired Gene Sequence for the Expression of PUFA Genes in Plant Expression Cassettes.

[0212] The Δ6-elongase Pp_PSE1 is first inserted into the first cassette in pUT123 via BstXI and XbaI. Then, the moss Δ6-desaturase (Pp_des6) is inserted via BamHI/NaeI into the second cassette and, finally, the Phaeodactylum Δ5-desaturase (Pt_des5) is inserted via BgIII/NcoI into the third cassette (see SEQ ID NO: 19). The triple construct is named pARA1. Taking into consideration sequence-specific restriction cleavage sites, further expression cassettes, as set out in table 3 and referred to as pARA2, pARA3 and pARA4, may be generated.

TABLE-US-00004 TABLE 3 Combinations of desaturases and elongases Gene plasmid Δ6-Desaturase Δ5-Desaturase Δ6-Elongase pARA1 Pp_des6 Pt_des5 Pp_PSE1 pARA2 Pt_des6 Pt_des5 Pp_PSE1 pARA3 Pt_des6 Ce_des5 Pp_PSE1 PARA4 Ce_des6 Ce_des5 Ce_PSE1 des5 = PUFA-specific Δ5-desaturase des6 = PUFA-specific Δ6-desaturase PSE = PUFA-specific Δ6-elongase Pt_des5 = Δ5-desaturase from Phaeodactylum tricornutum Pp_des6 or Pt_des6 = Δ6-desaturase from Physcomitrella patens or Phaeodactylum tricornutum Pp = Physcomitrella patens, Pt = Phaeodactylum tricornutum Pp_PSE1 = Δ6-elongase from Physcomitrella patens Pt_PSE1 = Δ6-elongase from Phaeodactylum tricornutum Ce_des5 = Δ5-desaturase from Caenorhabditis elegans (Genbank Acc. No. AF078796) Ce_des6 = Δ6-desaturase from Caenorhabditis elegans (Genbank Acc. No. AF031477, bases 11-1342) Ce_PSE1 = Δ6-elongase from Caenorhabditis elegans (Genbank Acc. No. AF244356, bases 1-867)

[0213] Further desaturases or elongase gene sequences may also be inserted into the expression cassettes of the type described, such as, for example, Genbank Acc. No. AF231981, NM_--013402, AF206662, AF268031, AF226273, AF110510 or AF110509.

iii.) Transfer of Expression Cassettes into Vectors for the Transformation of Agrobacterium tumefaciens and for the Transformation of Plants

[0214] The constructs thus generated were inserted into the binary vector pGPTV by means of AscI. For this purpose, the multiple cloning sequence was extended by an AscI cleavage site. For this purpose, the polylinker was synthesized de novo in the form of two double-stranded oligonucleotides, with an additional AscI DNA sequence being inserted. The oligonucleotide was inserted into the pGPTV vector by means of EcoRI and HindIII. The cloning techniques required are known to the skilled worker and may readily be found in the literature as described in example 1.

[0215] The nucleic acid sequences for Δ5-desaturase (SEQ ID NO: 13), Δ6-desaturase (SEQ ID NO: 9) and Δ6-elongase (SEQ ID NO: 11), which were used for the experiments described below, were the sequences from Physcomitrella patens and Phaeodactylum tricornutum. The corresponding amino acid sequences are the sequences SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. A vector which comprises all of the abovementioned genes is indicated in SEQ ID NO: 19. The corresponding amino acid sequences of the genes can be found in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

Example 2

Cloning and characterization of the ceLPLATs

a) Database Search

[0216] The ceLPLATs (=acyl-CoA:lysophospholipid acyltransferase from Caenorhabditis elegans) were identified by sequence comparisons with known LPA-ATs. The search was restricted to the nematode genome (Caenorhabditis elegans) with the aid of the BLAST-Psi algorithm (Altschul et al., J. Mol. Biol. 1990, 215: 403-410), since this organism synthesizes LCPUFAs. The probe employed in the sequence comparison was an LPAAT protein sequence from Mus musculus (MsLPAAT Accession No. NP_--061350). LPLAT catalyzes, by a reversible transferase reaction, the ATP-independent synthesis of acyl-CoAs from phospholipids with the aid of CoA as cofactor (Yamashita et al., J. Biol. Chem. 2001, 20: 26745-26752). Sequence comparisons enabled two putative ceLPLAT sequences to be identified (Accession No. T06E8.1 and F59F4.4). The identified sequences are most similar to each other and to MsLPAATs (FIG. 1). The alignment was generated using the Clustal program.

b) Cloning of the CeLPLATs

[0217] Primer pairs were synthesized on the basis of the ceLPLAT nucleic acid sequences (table 1) and the corresponding cDNAs were isolated from a C. elegans cDNA library by means of PCR processes. The respective primer pairs were selected so as to carry, apart from the start codon, the yeast consensus sequence for high-efficiency translation (Kozak, Cell 1986, 44:283-292). The LPLAT cDNAs were amplified in each case using 2 μl of cDNA-library solution as template, 200 μM dNTPs, 2.5 U of "proof-reading" pfu polymerase and 50 μmol of each primer in a total volume of 50 μl. The conditions for the PCR were as follows: first denaturation at 95° C. for 5 minutes, followed by 30 cycles at 94° C. for 30 seconds, 58° C. for one minute and 72° C. for 2 minutes, and a final extension step at 72° C. for 10 minutes. The sequence of the LPLAT cDNAs was confirmed by DNA sequencing.

TABLE-US-00005 TABLE 4 Nucleotide sequences of the PCR primers for cloning CeLPLATs Primer Nucleotide sequence 5' T06E8.1f* 5' ACATAATGGAGAACTTCTGGTCGATCGTC (SEQ ID NO: 50) 3' 3' T06E8.1r* 5' TTACTCAGATTTCTTCCCGTCTTT 3' (SEQ ID NO: 51) 5' F59F4.4f* 5' ACATAATGACCTTCCTAGCCATATTA 3' (SEQ ID NO: 52) 3' F59F4.4r* 5' TCAGATATTCAAATTGGCGGCTTC 3' (SEQ ID NO: 53) *f: forward, r: reverse

Example 3

Analysis of the Effect of the Recombinant Proteins on Production of the Desired Product

a) Possible Preparation Methods

[0218] The effect of genetic modification in fungi, algae, ciliates or, as described in the examples hereinabove, on the production of the polyunsaturated fatty acids in yeasts, or in plants may be determined by growing the modified microorganisms or the modified plant under suitable conditions (such as those described above) and studying the medium and/or the cellular components for increased production of the lipids or fatty acids. These analytical techniques are known to the skilled worker and comprise spectroscopy, thin layer chromatography, various types of staining methods, enzymic and microbiological methods and analytical chromatography such as high-performance liquid chromatography (see, for example, Ullmann, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol. 3, chapter III: "Product recovery and purification", pp. 469-714, VCH: Weinheim; Better, P. A., et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and Henry, J. D. (1988)

[0219] Biochemical Separations, in: Ullmann's Encyclopedia of Industrial Chemistry, vol. B3; chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

[0220] Apart from the abovementioned methods for detecting fatty acids in yeasts, plant lipids are extracted from plant material as described by Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96 (22):12935-12940, and Browse et al. (1986) Analytic Biochemistry 152:141-145. The qualitative and quantitative analysis of lipids or fatty acids is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307 S. (Oily Press Lipid Library; 1); "Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952) 16 (1977) under the title.: Progress in the Chemistry of Fats and Other Lipids CODEN.

[0221] Thus, fatty acids or triacylglycerol (=TAG, abbreviations indicated in brackets) may be analyzed, for example, by means of fatty acid methyl esters (=FAME), gas liquid chromatography-mass spectrometry (=GC-MS) or thin layer chromatography (TLC).

[0222] Unequivocal proof for the presence of fatty acid products may be obtained by means of analyzing recombinant organisms following standard analytical procedures: GC, GC-MS or TLC, as variously described by Christie and references therein (1997, in: Advances on Lipid Methodology, fourth ed.: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren [Gas chromatography-mass spectrometry methods], Lipide 33:343-353).

[0223] The plant material to be analyzed may for this purpose be disrupted either by sonification, glass milling, liquid nitrogen and grinding or via other applicable processes. After the material has been disrupted, it is then centrifuged. The sediment is then resuspended in distilled water, heated at 100° C. for 10 min, cooled on ice and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 h at 90° C., leading to hydrolyzed oil and lipid compounds which result in transmethylated lipids. These fatty acid methyl esters may then be extracted in petroleum ether and finally be subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 μm, 0.32 mm), with a temperature gradient of between 170° C. and 240° C. for 20 min and at 240° C. for 5 min. The identity of the resulting fatty acid methyl esters can be defined using standards available from commercial sources (i.e. Sigma).

[0224] In the case of fatty acids for which no standards are available, the identity may be shown via derivatization and subsequent GC-MS analysis. For example, the localization of triple-bond fatty acids is shown via GC-MS after derivatization with 4,4-dimethoxyoxazoline derivatives (Christie, 1998, see above).

b) Fatty Acid Analysis in Plants

[0225] Total fatty acids were extracted from plant seeds and analyzed by means of gas chromatography.

[0226] The seeds were taken up with 1% sodium methoxide in methanol and incubated at RT (approx. 22° C.) for 20 min. This was followed by washing with NaCl solution and taking up the FAMEs in 0.3 ml of heptane.

[0227] The samples were fractionated on a ZEBRON-ZB Wax capillary column (30 m, 0.32 mm, 0.25 μm; Phenomenex) in a Hewlett Packard 6850 gas chromatograph with flame ionization detector. The oven temperature was programmed from 70° C. (hold for 1 min) to 200° C. at a rate of 20° C./min, then to 250° C. (hold for 5 min) at a rate of 5° C./min and finally to 260° C. at a rate of 5° C./min. The carrier gas used was nitrogen (4.5 ml/min at 70° C.). The fatty acids were identified by comparison with retention times of FAME standards (SIGMA).

Example 4

Functional Characterization of CeLPLATs in Yeast

[0228] a) Heterologous Expression in Saccharomyces cerevisiae

[0229] To characterize the function of the C. elegans CeLPLATs, the open reading frames of the particular cDNAs were cloned downstream of the galactose-inducible GAL1 promoter of pYes2.1Topo, using the pYes2.1TOPO TA Expression Kit (Invitrogen), resulting in pYes2-T06E8.1 and pYes2-F59F4.4.

[0230] Since expression of the CeLPLATs should result in an efficient exchange of the acyl substrates, the double construct pESCLeu-PpD6-Pse1 which includes the open reading frames of a Δ6-desaturase (PpD6) and a Δ6-elongase (PSE1) from Physcomitrella patens (see DE 102 19 203) was also prepared. The nucleic acid sequence of said Δ6-desaturase (PpD6) and said Δ6-elongase (Pse1) are indicated in each case in SEQ ID NO: 9 and SEQ ID NO: 11. The corresponding amino acid sequences can be found in SEQ ID NO: 10 and SEQ ID NO: 12.

[0231] The Saccharomyces cerevisiae strains C13ABYS86 (protease-deficient) and INVSc1 were transformed simultaneously with the vectors pYes2-T06E8.1 and pESCLeu-PpD6-Pse1 and, respectively, pYes2-F59F4.4 and pESCLeu-PpD6-Pse1 by means of a modified PEG/lithium acetate protocol. The control used was a yeast which was transformed with the pESCLeu-PpD6-Pse1 vector and the empty vector pYes2. The transformed yeasts were selected on complete minimal medium (CMdum) agar plates containing 2% glucose but no uracil or leucine. After selection, 4 transformants, two pYes2-T06E8.1/pESCLeu-PpD6-Pse1 and two pYes2-F59F4.4/pESCLeu-PpD6-Pse1 and one pESCLeu-PpD6-Pse1/pYes2 were selected for further functional expression. The experiments described were also carried out in the yeast strain INVSc1.

[0232] In order to express the CeLPLATs, precultures of in each case 2 ml of CMdum liquid medium containing 2% (w/v) raffinose but no uracil or leucine were first inoculated with the selected transformants and incubated at 30° C., 200 rpm, for 2 days. 5 ml of CMdum liquid medium (without uracil and leucine) containing 2% raffinose, 1% (v/v) Tergitol NP-40 and 250 μM linoleic acid (18:2.sup.Δ9,12) or linolenic acid (18:3.sup.Δ9,12,15) were then inoculated with the precultures to an OD₆₀₀ of 0.08. Expression was induced at an OD₆₀₀ of 0.2-0.4 by adding 2% (w/v) galactose. The cultures were incubated at 20° C. for a further 48 h.

Fatty Acid Analysis

[0233] The yeast cells from the main cultures were harvested by centrifugation (100×g, 10 min, 20° C.) and washed with 100 mM NaHCO₃, pH 8.0 in order to remove residual medium and fatty acids. Fatty acid methyl esters (FAMEs) were prepared from the yeast cell sediments by acidic methanolysis. For this, the cell sediments were incubated with 2 ml of 1N methanolic sulfuric acid and 2% (v/v) dimethoxypropane at 80° C. for 1 h. Extraction of the FAMES was carried out by extracting twice with petroleum ether (PE). Nonderivatized fatty acids were removed by washing the organic phases in each case once with 2 ml of 100 mM NaHCO₃, pH 8.0 and 2 ml of distilled water. The PE phases were subsequently dried with Na₂SO₄, evaporated under argon and taken up in 100 μl of PE. The samples were separated on a DB-23 capillary column (30 m, 0.25 mm, 0.25 μm, Agilent) in a Hewlett-Packard 6850 gas chromatograph with flame ionization detector. The conditions for the GLC analysis were as follows: the oven temperature was programmed from 50° C. to 250° C. at a rate of 5° C./min and finally at 250° C. (hold) for 10 min.

[0234] The signals were identified by comparing the retention times with those of corresponding fatty acid standards (Sigma).

Acyl-CoA Analysis

[0235] The acyl-CoA analysis was carried out as described in Larson and Graham (2001; Plant Journal 25: 115-125).

Expression Analysis

[0236] FIGS. 2 A and B and FIGS. 3 A and B depict the fatty acid profiles of transgenic C13ABYS86 yeasts fed with 18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15, respectively. The substrates fed can be detected in large amounts in all transgenic yeasts. All four transgenic yeasts display synthesis of 18:3.sup.Δ6,9,12 and 20:3.sup.Δ8,11,14 and, respectively, 18:4.sup.Δ6,9,12,15 and 20:4.sup.Δ8,11,14,17, the products of the Δ6-desaturase and Δ6-elongase reactions, meaning that the genes PpD6 and Pse1 were able to be functionally expressed.

[0237] FIG. 2 depicts, as described above, the fatty acid profiles of transgenic C13ABYS86 S. cerevisiae cells. The fatty acid methyl esters were synthesized by acidic methanolysis of intact cells which had been transformed either with the pESCLeu-PpD6-Pse1/pYes2 (A) or with the pYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B) vectors. The yeasts were cultured in minimal medium in the presence of 18:2.sup.Δ9,12. The fatty acid methyl esters were subsequently analyzed by GLC.

[0238] In the control yeasts transformed with the pESCLeu-PpD6-Pse1/pYes2 vectors, the proportion of 20:3.sup.Δ8,11,14 to which 18:3.sup.Δ6,9,12 is elongated by Pse1 is substantially lower than in the yeasts which additionally express LPLAT T06E8.1. In fact, elongation of 18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15 was improved by 100-150% by additional expression of CeLPLAT (T06E8.1) (FIG. 4). This significant increase in the LCPUFA content can be explained only as follows: the exogenously fed fatty acids (18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15, respectively) are first incorporated into phospholipids and desaturated there by Δ6-desaturase to give 18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15. Only after reequilibration with the acyl-CoA pool, 18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15 can be elongated by the elongase to give 20:3.sup.Δ8,11,14- and 20:4.sup.Δ8,11,14,17-CoA, respectively and then incorporated again into the lipids. LPLAT T06E8.1 is capable of converting the Δ6-desaturated acyl groups very efficiently back to CoA thioesters. Interestingly, it was also possible to improve the elongation of the fed fatty acids 18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15. (FIGS. 2 A and B and FIGS. 3 A and B, respectively).

[0239] FIG. 3 indicates the fatty acid profiles of transgenic C13ABYS86 S. cerevisiae cells. Synthesis of the fatty acid methyl esters was carried out by acidic methanolysis of intact cells which had been transformed either with the vectors pESCLeu-PpD6-Pse1/pYes2 (A) or with the vectors pYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B). The yeasts were cultured in minimal medium in the presence of 18:3.sup.Δ9,12,15. The fatty acid methyl esters were subsequently analyzed via GLC.

[0240] In contrast, expression of a different CeLPLAT (F59F4.4) has no influence on elongation (FIG. 4). F59F4.4 evidently does not encode an LPLAT. Thus, not every putative LPLAT nucleic acid sequence is enzymatically active in the reaction found according to the invention.

[0241] FIG. 4 indicates the elongation of exogenously applied 18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15, following their endogenous Δ6-desaturation (data of FIGS. 2 and 3). The exogenously fed fatty acids are first incorporated into phospholipids and desaturated there to give 18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15. Only after reequilibration with the acyl-CoA pool can 18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15 be elongated by the elongase to give 20:3.sup.Δ8,11,14- and 20:4.sup.Δ8,11,14,17-CoA, respectively, and then incorporated again into the lipids. LPLAT T06E8.1 is capable of converting the Δ6-desaturated acyl groups efficiently back to CoA-thioesters.

[0242] These results show that CeLPLAT (T06E8.1) after coexpression with Δ6-desaturase and Δ6-elongase, leads to efficient production of C20-PUFAs. These results can be explained by the fact that CeLPLAT (T06E8.1) makes possible an efficient exchange of the newly synthesized fatty acids between lipids and the acyl-CoA pool (see FIG. 7).

[0243] FIG. 7 indicates the acyl-CoA composition of transgenic INVSc1 yeasts transformed with the pESCLeu PpD6Pse1/pYes2 (A) or pESCLeu-PpD6-Pse1/pYes2-T06E8.1 (B) vectors. The yeast cells were cultured in minimal medium without uracil and leucine in the presence of 250 μM 18:2.sup.Δ9,12. The acyl-CoA derivatives were analyzed via HPLC.

[0244] When using the yeast strain INVSc1 for coexpression of CeLPLAT (T06E8.1) together with PpD6 and Pse1, the following picture emerges: control yeasts expressing PpD6 and Pse1 comprise, as already shown when using the strain C13ABYS86, only small amounts of the elongation product (20:3.sup.Δ8,11,14, with 18:2 feed, and 20:4.sup.Δ8,11,14,17, with 18:3 feed; see FIGS. 5 A and 6 A, respectively). Additional expression of CeLPLAT (T06E8.1) results in a marked increase in these elongation products (see FIGS. 5 B and 6 B). Table 6 indicates that additional expression of CeLPLAT surprisingly causes an 8 fold increase in the 20:3.sup.Δ8,11,14 (with 18:2 feed) and, respectively, the 20:4.sup.Δ8,11,14,17 (with 18:3 feed) content. It is also revealed that C16:2.sup.Δ6,9 is also elongated more efficiently to give C18:2.sup.Δ6,9.

TABLE-US-00006 TABLE 5 Fatty acid composition (in mol %) of transgenic yeasts transformed with the pESCLeu PpD6Pse1/pYes2 (PpD6 Pse1) or pESCLeu-PpD6-Pse1/ pYes2-T06E8.1 (PpD6 Pse1 + T06E8) vectors. The yeast cells were cultured in minimal medium without uracil and leucine in the presence of 250 μM 18:2.sup.Δ9,12 or 18:3.sup.Δ9,12,15. The fatty acid methyl esters were obtained by acidic methanolysis of whole cells and analyzed via GLC. Each value indicates the average (n = 4) ± standard deviation. Feeding with Feeding with 250 μM 18:2^9,12 250 μM 18:3^9,12,15 Pp 6/Pse1 + Pp 6/Pse1 + Fatty acids Pp 6/Pse1 T06E8 Pp 6/Pse1 T06E8 16:0 15.31 ± 1.36 15.60 ± 1.36 12.20 ± 0.62 16.25 ± 1.85 16:1.sup.δ9 23.22 ± 2.16 15.80 ± 3.92 17.61 ± 1.05 14.58 ± 1.93 18:0 5.11 ± 0.63 7.98 ± 1.28 5.94 ± 0.71 7.52 ± 0.89 18:1.sup.δ9 15.09 ± 0.59 16.01 ± 2.53 15.62 ± 0.34 15.14 ± 2.61 18:1.sup.δ11 4.64 ± 1.09 11.80 ± 1.12 4.56 ± 0.18 13.07 ± 1.66 18:2.sup.δ9,12 28.72 ± 3.25 14.44 ± 1.61 -- -- 18:3.sup.δ6,9,12 3.77 ± 0.41 4.72 ± 0.72 -- -- 18:3.sup.δ9,12,15 -- -- 32.86 ± 1.20 14.14 ± 2.52 18:4.sup.δ6,9,12,15 -- -- 5.16 ± 1.04 3.31 ± 1.15 20:2.sup.δ11,14 2.12 ± 0.86 4.95 ± 4.71 -- -- 20:3.sup.δ8,11,14 1.03 ± 0.14 8.23 ± 1.59 -- -- 20:3.sup.δ11,14,17 -- -- 4.12 ± 1.54 6.95 ± 2.52 20:4.sup.δ8,11,14,17 -- -- 1.34 ± 0.28 8.70 ± 1.11

[0245] The fatty acid profile of transgenic INVSc1 S. cerevisiae cells can be found in FIG. 5. The fatty acid methyl esters were synthesized by acidic methanolysis of intact cells which had been transformed either with the pESCLeu-PpD6-Pse1/pYes2 (A) or with the pYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B) vectors. The yeasts were cultured in minimal medium in the presence of 18:2.sup.Δ9,12. The fatty acid methyl esters were subsequently analyzed via GLC.

[0246] FIG. 6 depicts the fatty acid profiles of transgenic INVSc1 S. cerevisiae cells. The fatty acid methyl esters were synthesized by acidic methanolysis of intact cells which had been transformed either with the pESCLeu-PpD6-Pse1/pYes2 (A) or with the pYes2-T06E8.1/pESCLeu-PpD6-Pse1 (B) vectors. The yeasts were cultured in minimal medium in the presence of 18:3.sup.Δ,12,15. The fatty acid methyl esters were subsequently analyzed via GLC.

[0247] A measure for the efficiency of LCPUFA biosynthesis in transgenic yeast is the quotient of the content of the desired Δ6-elongation product after Δ6-desaturation (20:3.sup.Δ8,11,14 and 20:4.sup.Δ8,11,14,17, respectively) to the content of fatty acid fed in (18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15, respectively). This quotient is 0.04 in INVSc1 control yeasts expressing PpD6 and Pse1, and 0.60 in yeasts expressing CeLPLAT in addition to PpD6 and Pse1. In other words: the content of desired Δ6-elongation product after Δ6-desaturation with coexpression of CeLPLAT is 60% of the content of the fatty acid fed in in each case. In control yeasts, this content is only approx. 4%, meaning a 15 fold increase in the efficiency of LCPUFA biosynthesis in transgenic yeast due to coexpression of LPLAT.

[0248] Interestingly, coexpression of CeLPLAT causes not only an increase in the elongation products mentioned, 20:3.sup.Δ8,11,14 and 20:4.sup.Δ8,11,14,17, also an increase in the 20:3.sup.Δ8,11,14:20:2.sup.Δ11,14 ratio and the 20:4.sup.Δ8,11,14,17:20:3.sup.Δ11,14,17 ratio, respectively. This means that, in the presence of LPLAT, Δ6-elongase preferably uses polyunsaturated fatty acids (18:3.sup.Δ6,9,12 and 18:4.sup.Δ6,9,12,15) as substrate, while no distinct substrate specificity is discernible in the absence of LPLAT (18:2.sup.Δ9,12 and 18:3.sup.Δ9,12,15 are also elongated). The reason for this may be protein-protein interactions between Δ6-elongase, Δ6-desaturase and LPLAT or posttranslational modifications (partial proteolysis, for example). This will also explain why the above-described rise in Δ6-elongation products with coexpression of Δ6-desaturase, Δ6-elongase and LPLAT is smaller when a protease-deficient yeast strain is used.

[0249] Acyl-CoA analyses of transgenic INVSc1 yeasts fed with 18:2.sup.Δ9,12 gave the following result: no 18:3.sup.Δ6,9,12-CoA and 20:3.sup.Δ8,11,14-CoA is detectable in control yeasts expressing PpD6 and Pse1, indicating that neither the substrate (18:3.sup.Δ6,9,12-CoA) nor the product (20:3.sup.Δ8,11,14-CoA) of Δ6-elongase is present in detectable amounts in control yeasts. This suggests that the transfer of 18:3.sup.Δ6,9,12 from membrane lipids into the acyl-CoA pool does not take place or does not take place correctly, meaning that there is hardly any substrate available for the Δ6-elongase present, and this in turn explains the low elongation product content in control yeasts. INVSc1 yeasts which express CeLPLAT in addition to PpD6 and Pse1 and which had been fed with 18:2.sup.Δ9,12 have substantial amounts of 20:3.sup.Δ8,11,14-CoA but not of 18:3.sup.Δ6,9,12-CoA. This indicates that LPLAT transfers 18:3.sup.Δ6,9,12 from the membrane lipids to the acyl-CoA pool very efficiently. 18:3.sup.Δ6,9,12-CoA is then elongated by Δ6-elongase so that 20:3.sup.Δ8,11,14-CoA but not any 18:3.sup.Δ6,9,12-CoA is detectable.

b) Functional Characterization of the CeLPLATs in Transgenic Plants

Expression of Functional CeLPLAT in Transgenic Plants

[0250] DE 102 19 203 describes transgenic plants whose seed oil comprises small amounts of ARA and EPA, due to seed-specific expression of functional genes coding for Δ6-desaturase, Δ6-elongase and Δ5-desaturase. The vector exploited for transformation of these plants can be found in SEQ ID NO: 19. In order to increase the content of these LCPUFAs, the gene CeLPLAT (T06E8.1) was additionally expressed in seeds in the transgenic plants mentioned.

[0251] For this purpose, the coding region of CeLPLAT was amplified via PCR.

[0252] Table 6 indicates the primers used for cloning another CeLPLAT clone into binary vectors.

TABLE-US-00007 TABLE 6 Nucleotide sequences of the PCR primers for cloning CeLPLAT (T06E8.1) into the binary vector pSUN3 Primer Nucleotide sequence ARe503f* (SEQ ID NO: 54) 5' TTAAGCGCGGCCGCATGGAGAACTTCTGGTCG 3' ARe504r* (SEQ ID NO: 55) 5' ACCTCGGCGGCCGCCCTTTTACTCAGATTTC 3' *f: forward, r: reverse

[0253] The PCR product was cloned into a pENTRY vector between USP promoter and OCS terminator. The expression cassette was then cloned into the binary pSUN300 vectors. The vector obtained was referred to as pSUN3CeLPLAT (FIG. 8). In addition, the CeLPLAT coding regions were amplified and cloned between LegB4 promoter and OCS terminator. This vector was referred to as pGPTVCeLPLAT (FIG. 9A).

[0254] In addition, the CeLPLAT coding regions were amplified via PCR and cloned between LegB4 promoter and OCS terminator. The PCR primers used for this were selected so as for an efficient Kozak sequence to be introduced into the PCR product. Moreover, the CeLPLAT DNA sequence was modified so as to adapt to the codon usage of higher plants.

[0255] The following primers were used for the PCR:

TABLE-US-00008 Forward primer (SEQ ID NO: 56): 5'-ACATAATGGAGAACTTCTGGTCTATTGTTGTGTTTTTTCTA-3' Reverse primer (SEQ ID NO: 57): 5'-CTAGCTAGCTTACTCAGATTTCTTCCCGTCTTTTGTTTCTC-3'

[0256] The PCR product was cloned into the cloning vector pCR Script and cloned via the restriction enzymes XmaI and SacI into the vector pGPTV LegB4-700. The resulting plasmid was referred to as pGPTV LegB4-700+T06E8.1 (FIG. 9A).

[0257] The same PCR product was in addition cloned into a multi-gene expression vector which already comprised the genes for a Phaeodactylum tricornutum delta-6-desaturase (SEQ ID NO: 32, amino acid sequence SEQ ID NO: 33) and a P. patens delta-6-elongase. The resulting plasmid was referred to as pGPTV USP/OCS-1,2,3 PSE1(Pp)+D6-Des(Pt)+2AT (T06E8-1) (FIG. 9B). The sequences of the vector and of the genes can be found in SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37. The Phaeodactylum tricornutum Δ6-desaturase extends from nucleotide 4554 to 5987 in SEQ ID NO: 34. The Physcomitrella patens Δ6-elongase extends from nucleotide 1026 to 1898 and that of Caenorhabditis elegans LPLAT extends from nucleotide 2805 to 3653 in SEQ ID NO: 34.

[0258] Tobacco plants were cotransformed with the pSUN3CeLPLAT vector and the vector described in DE 102 19 203 and SEQ ID NO: 19, which comprises genes coding for Δ6-desaturase, Δ6-elongase and Δ5-desaturase, with transgenic plants being selected using kanamycin.

[0259] Tobacco plants were moreover transformed with the pGPTV USP/OCS-1,2,3 PSE1(Pp)+D6-Des(Pt)+2AT (T06E8-1) vector [see SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37].

[0260] Linseed was transformed with the pSUN3CeLPLAT vector. The resulting transgenic plants were crossed with those transgenic linseed plants which already comprised small amounts of ARA and EPA, owing to functional gene expression of Δ6-desaturase, Δ6-elongase and Δ5-desaturase.

[0261] Linseed was furthermore transformed with the pGPTV LegB4-700+T06E8.1 vector. The resulting transgenic plants were crossed with those transgenic linseed plants which already comprised small amounts of ARA and EPA, owing to functional expression of Δ6-desaturase, Δ6-elongase and Δ5-desaturase.

[0262] The seeds of transgenic tobacco and linseed plants were, as described hereinbefore [example 3b)], studied for increased LCPUFAs contents.

[0263] The function of acyl-CoA:lysophopholipid acyltransferase (LPLAT) can be deduced from the studies presented herein as depicted in FIG. 10. The biosynthetic pathway of LCPUFAS is thus as follows.

[0264] Desaturases catalyze the introduction of double bonds into lipid-coupled fatty acids (sn2-acyl-phosphatidylcholine), while the elongases exclusively catalyze the elongation of coenzyme A-esterified fatty acids (acyl-CoAs). According to this mechanism, the alternating action of desaturases and elongases requires continuous exchange of acyl substrates between phospholipids and acyl-CoA pool and thus the existence of an additional activity which converts the acyl substrates to the substrate form required in each case, i.e. lipids (for desaturases) or CoA thioesters (for elongases). This exchange between acyl-CoA pool and phospholipids is made possible by LCPUFA-specific LPLAT. The biosynthesis of ARA (A) takes place analogously to that of EPA (B), but with the difference that, in the case of EPA, a Δ15-desaturation takes place upstream of the Δ6-desaturation so that α18:3-PC acts as a substrate for Δ6-desaturase. The biosynthesis of DHA requires a further exchange between phospholipids and acyl-CoA pool via LPLAT: 20:5.sup.Δ5,8,11,14,17 is transferred from the phospholipids pool to the CoA pool and, after Δ5-elongation, 22:5.sup.Δ7,10,13,16,19 is transferred from the CoA pool to the phospholipids pool and finally converted by Δ4-desaturase to give DHA. The same applies to the exchange in the biosynthetic pathway using Δ8-desaturase, Δ9-elongase and Δ5-desaturase.

EQUIVALENTS

[0265] Many equivalents of the specific embodiments of the invention described herein can be identified or found by the skilled worker by using merely routine experiments. These equivalents are intended to be within the scope of the patent claims.

Sequence CWU 1

1

591849DNACaenorhabditis elegansCDS(1)..(849)Acyl-CoAlysophospholipid acyltransferase 1atg gag aac ttc tgg tcg atc gtc gtg ttt ttt cta ctc tca att ctc 48Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 ttc att tta tat aac ata tcg aca gta tgc cac tac tat atg cgg att 96Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 tcg ttt tat tac ttc aca att tta ttg cat gga atg gaa gtt tgt gtt 144Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 aca atg atc cct tct tgg cta aat ggg aag ggt gct gat tac gtg ttt 192Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 cac tcg ttt ttc tat tgg tgt aaa tgg act ggt gtt cat aca aca gtc 240His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 tat gga tat gaa aaa aca caa gtt gaa ggt ccg gct gta gtt att tgt 288Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 aat cat cag agt tct ctc gac att cta tcg atg gca tca atc tgg ccg 336Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 aag aat tgt gtt gta atg atg aaa cga att ctt gcc tat gtt cca ttc 384Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 ttc aat ctc gga gcc tac ttt tcc aac aca atc ttc atc gat cga tat 432Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 aac cgt gaa cgt gcg atg gct tca gtt gat tat tgt gca tct gaa atg 480Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 aag aac aga aat ctt aaa ctt tgg gta ttt ccg gaa gga aca aga aat 528Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 cgt gaa gga ggg ttc att cca ttc aag aaa gga gca ttc aat att gca 576Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 gtt cgt gcg cag att ccc att att cca gtt gta ttc tca gac tat cgg 624Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 gat ttc tac tca aag cca ggc cga tat ttc aag aat gat gga gaa gtt 672Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 gtt att cga gtt ctg gat gcg att cca aca aaa ggg ctc act ctt gat 720Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 gac gtc agc gag ttg tct gat atg tgt cgg gac gtt atg ttg gca gcc 768Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 tat aag gaa gtt act cta gaa gct cag caa cga aat gcg aca cgg cgt 816Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 gga gaa aca aaa gac ggg aag aaa tct gag taa 849Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 2282PRTCaenorhabditis elegans 2Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 3849DNACaenorhabditis elegansCDS(1)..(849)Acyl-CoAlysophospholipid acyltransferase 3atg gag aac ttc tgg tcg atc gtc gtg ttt ttt cta ctc tca att ctc 48Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 ttc att tta tat aac ata tcg aca gta tgc cac tac tat atg cgg att 96Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 tcg ttt tat tac ttc aca att tta ttg cat gga atg gaa gtt tgt gtt 144Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 aca atg atc cct tct tgg cta aat ggg aag ggt gct gat tac gtg ttt 192Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 cac tcg ttt ttc tat tgg tgt aaa tgg act ggt gtt cat aca aca gtc 240His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 tat gga tat gaa aaa aca caa gtt gaa ggt ccg gct gta gtt att tgt 288Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 aat cat cag agt tct ctc gac att cta tcg atg gca tca atc tgg ccg 336Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 aag aat tgt gtt gta atg atg aaa cga att ctt gcc tat gtt cca ttc 384Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 ttc aat ctc gga gcc tac ttt tcc aac aca atc ttc atc gat cga tat 432Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 aac cgt gaa cgt gcg atg gct tca gtt gat tat tgt gca tct gaa atg 480Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 aag aac aga aat ctt aaa ctt tgg gta tct ccg gaa gga aca aga aat 528Lys Asn Arg Asn Leu Lys Leu Trp Val Ser Pro Glu Gly Thr Arg Asn 165 170 175 cgt gaa gga ggg ttc att cca ttc aag aaa gga gca ttc aat att gca 576Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 gtt cgt gcg cag att ccc att att cca gtt gta ttc tca gac tat cgg 624Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 gat ttc tac tca aag cca ggc cga tat ttc aag aat gat gga gaa gtt 672Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 gtt att cga gtt ctg gat gcg att cca aca aaa ggg ctc act ctt gat 720Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 gac gtc agc gag ttg tct gat atg tgt cgg gac gtt atg ttg gca gcc 768Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 tat aag gaa gtt act cta gaa gct cag caa cga aat gcg aca cgg cgt 816Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 gga gaa aca aaa gac ggg aag aaa tct gag taa 849Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 4282PRTCaenorhabditis elegans 4Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 Lys Asn Arg Asn Leu Lys Leu Trp Val Ser Pro Glu Gly Thr Arg Asn 165 170 175 Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 5849DNACaenorhabditis elegansCDS(1)..(849)Acyl-CoAlysophospholipid acyltransferase 5atg gag aac ttc tgg tcg atc gtc gtg ttt ttt cta ctc tca att ctc 48Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 ttc att tta tat aac ata tcg aca gta tgc cac tac tat gtg cgg att 96Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Val Arg Ile 20 25 30 tcg ttt tat tac ttc aca att tta ttg cat gga atg gaa gtt tgt gtt 144Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 aca atg atc cct tct tgg cta aat ggg aag ggt gct gat tac gtg ttt 192Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 cac tcg ttt ttc tat tgg tgt aaa tgg act ggt gtt cat aca aca gtc 240His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 tat gga tat gaa aaa aca caa gtt gaa ggt ccg gct gta gtt att tgt 288Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 aat cat cag agt tct ctc gac att cta tcg atg gca tca atc tgg ccg 336Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 aag aat tgt gtt gta atg atg aaa cga att ctt gcc tat gtt cca ttc 384Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 ttc aat ctc gga gcc tac ttt tcc aac aca atc ttc atc gat cga tat 432Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 aac cgt gaa cgt gcg atg gct tca gtt gat tat tgt gca tct gaa atg 480Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 aag aac aga aat ctt aaa ctt tgg gta ttt ccg gaa gga aca aga aat 528Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 cgt gaa gga ggg ttc att cca ttc aag aaa gga gca ttc aat att gca 576Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 gtt cgt gcg cag att ccc att att cca gtt gta ttc tca gac tat cgg 624Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 gat ttc tac tca aag cca ggc cga tat ttc aag aat gat gga gaa gtt 672Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 gtt att cga gtt ctg gat gcg att cca aca aaa ggg ctc act ctt gat 720Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 gac gtc agc gag ttg tct gat atg tgt cgg gac gtt atg ttg gca gcc 768Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 tat aag gaa gtt act cta gaa gct cag caa cga aat gcg aca cgg cgt 816Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 gga gaa aca aaa gac ggg aag aaa tct gag taa 849Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 6282PRTCaenorhabditis elegans 6Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Val Arg Ile 20 25 30 Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210

215 220 Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 7849DNACaenorhabditis elegansCDS(1)..(849)Acyl-CoAlysophospholipid acyltransferase 7atg gag aac ttc tgg tcg atc gtc gtg ttt ttt cta ctc tca att ctc 48Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 ttc att tta tat aac ata tcg aca gta tgc cac tac tat atg cgg att 96Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 tcg ttt tat tac ttc aca att tta ttg cat gga atg gaa gtt tgt gtt 144Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 aca atg atc cct tct tgg cta aat ggg aag ggt gct gat tac gtg ttt 192Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 cac tcg ttt ttc tat tgg tgt aaa tgg act ggt gtt cat aca aca gtc 240His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 tat gga tat gaa aaa aca caa gtt gaa ggt ccg gcc gta gtt att tgt 288Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 aat cat cag ggt tct ctc gac att cta tcg atg gca tca atc tgg ccg 336Asn His Gln Gly Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 aag aat tgt gtt gta atg atg aaa cga att ctt gcc tat gtt cca ttc 384Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 ttc aat ctc gga gcc tac ttt tcc aac aca atc ttc atc gat cga tat 432Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 aac cgt gaa cgt gcg atg gct tca gtt gat tat tgt gca tct gaa atg 480Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 aag aac aga aat ctt aaa ctt tgg gta ttt ccg gaa gga aca aga aat 528Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 cgt gaa gga ggg ttc att cca ttc aag aaa gga gca ttc aat att gca 576Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 gtt cgt gcg cag att ccc att att cca gtt gta ttc tca gac tat cgg 624Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 gat ttc tac tca aag cca ggc cga tat ttc aag aat gat gga gaa gtt 672Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 gtt att cga gtt ctg gat gcg att cca aca aaa ggg ctc act ctt gat 720Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 gac gtc agc gag ttg tct gat atg tgt cgg gac gtt atg ttg gca gcc 768Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 tat aag gaa gtt act cta gaa gct cag caa cga aat gcg aca cgg cgt 816Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 gga gaa aca aaa gac ggg aag aaa tct gag taa 849Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 8282PRTCaenorhabditis elegans 8Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 Asn His Gln Gly Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 91578DNAPhyscomitrella patensCDS(1)..(1578)Delta-6-desaturase 9atg gta ttc gcg ggc ggt gga ctt cag cag ggc tct ctc gaa gaa aac 48Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 5 10 15 atc gac gtc gag cac att gcc agt atg tct ctc ttc agc gac ttc ttc 96Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 20 25 30 agt tat gtg tct tca act gtt ggt tcg tgg agc gta cac agt ata caa 144Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 35 40 45 cct ttg aag cgc ctg acg agt aag aag cgt gtt tcg gaa agc gct gcc 192Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 50 55 60 gtg caa tgt ata tca gct gaa gtt cag aga aat tcg agt acc cag gga 240Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 65 70 75 80 act gcg gag gca ctc gca gaa tca gtc gtg aag ccc acg aga cga agg 288Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg 85 90 95 tca tct cag tgg aag aag tcg aca cac ccc cta tca gaa gta gca gta 336Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val 100 105 110 cac aac aag cca agc gat tgc tgg att gtt gta aaa aac aag gtg tat 384His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr 115 120 125 gat gtt tcc aat ttt gcg gac gag cat ccc gga gga tca gtt att agt 432Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser 130 135 140 act tat ttt gga cga gac ggc aca gat gtt ttc tct agt ttt cat gca 480Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala 145 150 155 160 gct tct aca tgg aaa att ctt caa gac ttt tac att ggt gac gtg gag 528Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu 165 170 175 agg gtg gag ccg act cca gag ctg ctg aaa gat ttc cga gaa atg aga 576Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe Arg Glu Met Arg 180 185 190 gct ctt ttc ctg agg gag caa ctt ttc aaa agt tcg aaa ttg tac tat 624Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr 195 200 205 gtt atg aag ctg ctc acg aat gtt gct att ttt gct gcg agc att gca 672Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe Ala Ala Ser Ile Ala 210 215 220 ata ata tgt tgg agc aag act att tca gcg gtt ttg gct tca gct tgt 720Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val Leu Ala Ser Ala Cys 225 230 235 240 atg atg gct ctg tgt ttc caa cag tgc gga tgg cta tcc cat gat ttt 768Met Met Ala Leu Cys Phe Gln Gln Cys Gly Trp Leu Ser His Asp Phe 245 250 255 ctc cac aat cag gtg ttt gag aca cgc tgg ctt aat gaa gtt gtc ggg 816Leu His Asn Gln Val Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 tat gtg atc ggc aac gcc gtt ctg ggg ttt agt aca ggg tgg tgg aag 864Tyr Val Ile Gly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 gag aag cat aac ctt cat cat gct gct cca aat gaa tgc gat cag act 912Glu Lys His Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 tac caa cca att gat gaa gat att gat act ctc ccc ctc att gcc tgg 960Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315 320 agc aag gac ata ctg gcc aca gtt gag aat aag aca ttc ttg cga atc 1008Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325 330 335 ctc caa tac cag cat ctg ttc ttc atg ggt ctg tta ttt ttc gcc cgt 1056Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg 340 345 350 ggt agt tgg ctc ttt tgg agc tgg aga tat acc tct aca gca gtg ctc 1104Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu 355 360 365 tca cct gtc gac agg ttg ttg gag aag gga act gtt ctg ttt cac tac 1152Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr 370 375 380 ttt tgg ttc gtc ggg aca gcg tgc tat ctt ctc cct ggt tgg aag cca 1200Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro 385 390 395 400 tta gta tgg atg gcg gtg act gag ctc atg tcc ggc atg ctg ctg ggc 1248Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met Leu Leu Gly 405 410 415 ttt gta ttt gta ctt agc cac aat ggg atg gag gtt tat aat tcg tct 1296Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val Tyr Asn Ser Ser 420 425 430 aaa gaa ttc gtg agt gca cag atc gta tcc aca cgg gat atc aaa gga 1344Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr Arg Asp Ile Lys Gly 435 440 445 aac ata ttc aac gac tgg ttc act ggt ggc ctt aac agg caa ata gag 1392Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu 450 455 460 cat cat ctt ttc cca aca atg ccc agg cat aat tta aac aaa ata gca 1440His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Lys Ile Ala 465 470 475 480 cct aga gtg gag gtg ttc tgt aag aaa cac ggt ctg gtg tac gaa gac 1488Pro Arg Val Glu Val Phe Cys Lys Lys His Gly Leu Val Tyr Glu Asp 485 490 495 gta tct att gct acc ggc act tgc aag gtt ttg aaa gca ttg aag gaa 1536Val Ser Ile Ala Thr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 gtc gcg gag gct gcg gca gag cag cat gct acc acc agt taa 1578Val Ala Glu Ala Ala Ala Glu Gln His Ala Thr Thr Ser 515 520 525 10525PRTPhyscomitrella patens 10Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 5 10 15 Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 20 25 30 Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 35 40 45 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 50 55 60 Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 65 70 75 80 Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg 85 90 95 Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val 100 105 110 His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr 115 120 125 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser 130 135 140 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala 145 150 155 160 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu 165 170 175 Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe Arg Glu Met Arg 180 185 190 Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr 195 200 205 Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe Ala Ala Ser Ile Ala 210 215 220 Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val Leu Ala Ser Ala Cys 225 230 235 240 Met Met Ala Leu Cys Phe Gln Gln Cys Gly Trp Leu Ser His Asp Phe 245 250 255 Leu His Asn Gln Val Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 Tyr Val Ile Gly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 Glu Lys His Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315 320 Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325 330 335 Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg 340 345 350 Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu 355 360 365 Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr 370 375 380 Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro 385 390 395 400 Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met Leu Leu Gly 405 410 415 Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val Tyr Asn Ser Ser 420 425 430 Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr Arg Asp Ile Lys Gly 435 440 445 Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu 450 455 460 His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Lys Ile Ala 465 470 475 480 Pro Arg Val Glu Val Phe Cys Lys Lys His Gly Leu Val Tyr Glu Asp 485 490 495 Val Ser Ile Ala Thr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 Val Ala Glu Ala Ala Ala Glu Gln His Ala Thr Thr Ser 515 520 525 111192DNAPhyscomitrella patensCDS(58)..(930)Delta-6-elongase 11ctgcttcgtc tcatcttggg ggtgtgattc

gggagtgggt tgagttggtg gagcgca 57atg gag gtc gtg gag aga ttc tac ggt gag ttg gat ggg aag gtc tcg 105Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser 1 5 10 15 cag ggc gtg aat gca ttg ctg ggt agt ttt ggg gtg gag ttg acg gat 153Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp 20 25 30 acg ccc act acc aaa ggc ttg ccc ctc gtt gac agt ccc aca ccc atc 201Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 40 45 gtc ctc ggt gtt tct gta tac ttg act att gtc att gga ggg ctt ttg 249Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 50 55 60 tgg ata aag gcc agg gat ctg aaa ccg cgc gcc tcg gag cca ttt ttg 297Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 ctc caa gct ttg gtg ctt gtg cac aac ctg ttc tgt ttt gcg ctc agt 345Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 ctg tat atg tgc gtg ggc atc gct tat cag gct att acc tgg cgg tac 393Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 tct ctc tgg ggc aat gca tac aat cct aaa cat aaa gag atg gcg att 441Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 ctg gta tac ttg ttc tac atg tct aag tac gtg gaa ttc atg gat acc 489Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 gtt atc atg ata ctg aag cgc agc acc agg caa ata agc ttc ctc cac 537Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155 160 gtt tat cat cat tct tca att tcc ctc att tgg tgg gct att gct cat 585Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165 170 175 cac gct cct ggc ggt gaa gca tat tgg tct gcg gct ctg aac tca gga 633His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190 gtg cat gtt ctc atg tat gcg tat tac ttc ttg gct gcc tgc ctt cga 681Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200 205 agt agc cca aag tta aaa aat aag tac ctt ttt tgg ggc agg tac ttg 729Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210 215 220 aca caa ttc caa atg ttc cag ttt atg ctg aac tta gtg cag gct tac 777Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr 225 230 235 240 tac gac atg aaa acg aat gcg cca tat cca caa tgg ctg atc aag att 825Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile 245 250 255 ttg ttc tac tac atg atc tcg ttg ctg ttt ctt ttc ggc aat ttt tac 873Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr 260 265 270 gta caa aaa tac atc aaa ccc tct gac gga aag caa aag gga gct aaa 921Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 275 280 285 act gag tga gctgtatcaa gccatagaaa ctctattatg ttagaacctg 970Thr Glu 290 aagttggtgc tttcttatct ccacttatct tttaagcagc atcagttttg aaatgatgtg 1030tgggcgtggt ctgcaagtag tcatcaatat aatcggcctg agcacttcag atggattgtt 1090agaacatgag taaaagcggt tattacggtg tttattttgt accaaatcac cgcacgggtg 1150aattgaaata tttcagattt gatcaatttc atctgaaaaa aa 119212290PRTPhyscomitrella patens 12Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser 1 5 10 15 Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp 20 25 30 Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 40 45 Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 50 55 60 Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155 160 Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165 170 175 His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190 Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200 205 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210 215 220 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr 225 230 235 240 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile 245 250 255 Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr 260 265 270 Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 275 280 285 Thr Glu 290 131410DNAPhaeodactylum tricornutumCDS(1)..(1410)Delta-5-desaturase 13atg gct ccg gat gcg gat aag ctt cga caa cgc cag acg act gcg gta 48Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 gcg aag cac aat gct gct acc ata tcg acg cag gaa cgc ctt tgc agt 96Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 ctg tct tcg ctc aaa ggc gaa gaa gtc tgc atc gac gga atc atc tat 144Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 gac ctc caa tca ttc gat cat ccc ggg ggt gaa acg atc aaa atg ttt 192Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 ggt ggc aac gat gtc act gta cag tac aag atg att cac ccg tac cat 240Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 acc gag aag cat ttg gaa aag atg aag cgt gtc ggc aag gtg acg gat 288Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 ttc gtc tgc gag tac aag ttc gat acc gaa ttt gaa cgc gaa atc aaa 336Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 cga gaa gtc ttc aag att gtg cga cga ggc aag gat ttc ggt act ttg 384Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 gga tgg ttc ttc cgt gcg ttt tgc tac att gcc att ttc ttc tac ctg 432Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 cag tac cat tgg gtc acc acg gga acc tct tgg ctg ctg gcc gtg gcc 480Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160 tac gga atc tcc caa gcg atg att ggc atg aat gtc cag cac gat gcc 528Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170 175 aac cac ggg gcc acc tcc aag cgt ccc tgg gtc aac gac atg cta ggc 576Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 ctc ggt gcg gat ttt att ggt ggt tcc aag tgg ctc tgg cag gaa caa 624Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 cac tgg acc cac cac gct tac acc aat cac gcc gag atg gat ccc gat 672His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 agc ttt ggt gcc gaa cca atg ctc cta ttc aac gac tat ccc ttg gat 720Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 cat ccc gct cgt acc tgg cta cat cgc ttt caa gca ttc ttt tac atg 768His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255 ccc gtc ttg gct gga tac tgg ttg tcc gct gtc ttc aat cca caa att 816Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265 270 ctt gac ctc cag caa cgc ggc gca ctt tcc gtc ggt atc cgt ctc gac 864Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275 280 285 aac gct ttc att cac tcg cga cgc aag tat gcg gtt ttc tgg cgg gct 912Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 290 295 300 gtg tac att gcg gtg aac gtg att gct ccg ttt tac aca aac tcc ggc 960Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 ctc gaa tgg tcc tgg cgt gtc ttt gga aac atc atg ctc atg ggt gtg 1008Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 gcg gaa tcg ctc gcg ctg gcg gtc ctg ttt tcg ttg tcg cac aat ttc 1056Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 gaa tcc gcg gat cgc gat ccg acc gcc cca ctg aaa aag acg gga gaa 1104Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 cca gtc gac tgg ttc aag aca cag gtc gaa act tcc tgc act tac ggt 1152Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 gga ttc ctt tcc ggt tgc ttc acg gga ggt ctc aac ttt cag gtt gaa 1200Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400 cac cac ttg ttc cca cgc atg agc agc gct tgg tat ccc tac att gcc 1248His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 ccc aag gtc cgc gaa att tgc gcc aaa cac ggc gtc cac tac gcc tac 1296Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 tac ccg tgg atc cac caa aac ttt ctc tcc acc gtc cgc tac atg cac 1344Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 gcg gcc ggg acc ggt gcc aac tgg cgc cag atg gcc aga gaa aat ccc 1392Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 ttg acc gga cgg gcg taa 1410Leu Thr Gly Arg Ala 465 14469PRTPhaeodactylum tricornutum 14Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170 175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255 Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275 280 285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 290 295 300 Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400 His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 Leu Thr Gly Arg Ala 465 153598DNAArtificial sequenceThis sequence is a plant promoter-terminator expression cassette in vector pUC19 15tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccg agctcctcga 420gcaaatttac acattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720gcaatgctgc atggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780taccacacaa gatttgaggt gcatgaacgt cacgtggaca

aaaggtttag taatttttca 840agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020atataatgag gattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080taatttcttc atagccagcc caccgcggtg ggcggccgcc tgcagtctag aaggcctcct 1140gctttaatga gatatgcgag acgcctatga tcgcatgata tttgctttca attctgttgt 1200gcacgttgta aaaaacctga gcatgtgtag ctcagatcct taccgccggt ttcggttcat 1260tctaatgaat atatcacccg ttactatcgt atttttatga ataatattct ccgttcaatt 1320tactgattgt ccgtcgacga attcgagctc ggcgcgccaa gcttggcgta atcatggtca 1380tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 1440agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 1500cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 1560caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 1620tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 1680cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 1740aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 1800gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 1860agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 1920cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 1980cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 2040ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 2100gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 2160tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 2220acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 2280tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 2340attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 2400gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 2460ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 2520taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 2580ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 2640ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 2700gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 2760ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 2820gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 2880tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 2940atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 3000gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 3060tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 3120atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 3180agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 3240ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 3300tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 3360aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 3420tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 3480aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 3540accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 3598163590DNAArtificial sequenceThis sequence is a plant promoter-terminator expression cassette in vector pUC19 16tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccg agctcctcga 420gcaaatttac acattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720gcaatgctgc atggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttag taatttttca 840agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020atataatgag gattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080taatttcttc atagccagcg gatccgatat cgggcccgct agcgttaacc ctgctttaat 1140gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg 1200taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga 1260atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt 1320gtccgtcgac gaattcgagc tcggcgcgcc aagcttggcg taatcatggt catagctgtt 1380tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 1440gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 1500gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 1560ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 1620ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 1680cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 1740gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 1800tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 1860ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 1920atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 1980gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 2040tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 2100cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 2160cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 2220tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 2280cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 2340cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 2400gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 2460gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 2520gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 2580ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 2640atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 2700agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 2760ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 2820tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 2880ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 2940caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 3000gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 3060atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 3120accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 3180aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 3240gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 3300tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 3360aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 3420ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 3480aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat 3540tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 3590173584DNAArtificial sequenceThis sequence is a plant promoter-terminator expression cassette in vector pUC19 17tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccg agctcctcga 420gcaaatttac acattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720gcaatgctgc atggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttag taatttttca 840agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020atataatgag gattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080taatttcttc atagccagca gatctgccgg catcgatccc gggccatggc ctgctttaat 1140gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg 1200taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga 1260atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt 1320gtccgtcgac gagctcggcg cgccaagctt ggcgtaatca tggtcatagc tgtttcctgt 1380gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 1440agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 1500tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 1560aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 1620cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 1680atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 1740taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1800aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1860tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1920gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1980cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 2040cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 2100atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 2160tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 2220ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 2280acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 2340aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 2400aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 2460tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 2520cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 2580catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 2640ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 2700aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 2760ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2820caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2880attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2940agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 3000actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 3060ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 3120ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 3180gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 3240atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 3300cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 3360gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 3420gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 3480ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 3540gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 3584184507DNAArtificial sequenceThis sequence is a plant promoter-terminator expression cassette in vector pUC19 18tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cggcgcgccg agctcctcga 420gcaaatttac acattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720gcaatgctgc atggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttag taatttttca 840agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020atataatgag gattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080taatttcttc atagccagcc caccgcggtg ggcggccgcc tgcagtctag aaggcctcct 1140gctttaatga gatatgcgag acgcctatga tcgcatgata tttgctttca attctgttgt 1200gcacgttgta aaaaacctga gcatgtgtag ctcagatcct taccgccggt ttcggttcat 1260tctaatgaat atatcacccg ttactatcgt atttttatga ataatattct ccgttcaatt 1320tactgattgt ccgtcgagca aatttacaca ttgccactaa acgtctaaac ccttgtaatt 1380tgtttttgtt ttactatgtg tgttatgtat ttgatttgcg ataaattttt atatttggta 1440ctaaatttat aacacctttt atgctaacgt ttgccaacac ttagcaattt gcaagttgat 1500taattgattc taaattattt ttgtcttcta aatacatata ctaatcaact ggaaatgtaa 1560atatttgcta atatttctac tataggagaa ttaaagtgag tgaatatggt accacaaggt 1620ttggagattt aattgttgca atgctgcatg gatggcatat acaccaaaca ttcaataatt 1680cttgaggata ataatggtac cacacaagat ttgaggtgca tgaacgtcac gtggacaaaa 1740ggtttagtaa tttttcaaga caacaatgtt accacacaca agttttgagg tgcatgcatg 1800gatgccctgt ggaaagttta aaaatatttt ggaaatgatt tgcatggaag ccatgtgtaa 1860aaccatgaca tccacttgga ggatgcaata atgaagaaaa ctacaaattt acatgcaact 1920agttatgcat gtagtctata taatgaggat tttgcaatac tttcattcat acacactcac 1980taagttttac acgattataa tttcttcata gccagcggat ccgatatcgg gcccgctagc 2040gttaaccctg ctttaatgag atatgcgaga cgcctatgat cgcatgatat ttgctttcaa 2100ttctgttgtg cacgttgtaa aaaacctgag catgtgtagc tcagatcctt accgccggtt 2160tcggttcatt ctaatgaata tatcacccgt tactatcgta tttttatgaa taatattctc 2220cgttcaattt actgattgtc cgtcgacgaa ttcgagctcg gcgcgccaag cttggcgtaa 2280tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 2340cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 2400attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 2460tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 2520ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 2580gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2640ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2700cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2760ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2820accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2880catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2940gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 3000tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 3060agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 3120actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 3180gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 3240aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3300gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3360aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3420atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3480gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3540atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3600ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3660cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3720agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3780cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3840tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3900agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3960gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 4020gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 4080ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 4140tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 4200tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 4260gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4320caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4380atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4440gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4500tttcgtc 45071917752DNAArtificial sequenceConstruct comprising sequences encoding Delta-6-elongase of Physcomitrella patens, Delta-6-desaturase of Physcomitrella patens, and Delta-5-desaturase of Phaeodactylum

tricornutum 19gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagccca ccgcggtgga aa atg gag gtc gtg gag aga ttc tac ggt gag 11572 Met Glu Val Val Glu Arg Phe Tyr Gly Glu 1 5 10 ttg gat ggg aag gtc tcg cag ggc gtg aat gca ttg ctg ggt agt ttt 11620Leu Asp Gly Lys Val Ser Gln Gly Val Asn Ala Leu Leu Gly Ser Phe 15 20 25 ggg gtg gag ttg acg gat acg ccc act acc aaa ggc ttg ccc ctc gtt 11668Gly Val Glu Leu Thr Asp Thr Pro Thr Thr Lys Gly Leu Pro Leu Val 30 35 40 gac agt ccc aca ccc atc gtc ctc ggt gtt tct gta tac ttg act att 11716Asp Ser Pro Thr Pro Ile Val Leu Gly Val Ser Val Tyr Leu Thr Ile 45 50 55 gtc att gga ggg ctt ttg tgg ata aag gcc agg gat ctg aaa ccg cgc 11764Val Ile Gly Gly Leu Leu Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg 60 65 70 gcc tcg gag cca ttt ttg ctc caa gct ttg gtg ctt gtg cac aac ctg 11812Ala Ser Glu Pro Phe Leu Leu Gln Ala Leu Val Leu Val His Asn Leu 75 80 85 90 ttc tgt ttt gcg ctc agt ctg tat atg tgc gtg ggc atc gct tat cag 11860Phe Cys Phe Ala Leu Ser Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln 95 100 105 gct att acc tgg cgg tac tct ctc tgg ggc aat gca tac aat cct aaa 11908Ala Ile Thr Trp Arg Tyr Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys 110 115 120 cat aaa gag atg gcg att ctg gta tac ttg ttc tac atg tct aag tac 11956His Lys Glu Met Ala Ile Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr 125 130 135 gtg gaa ttc atg gat acc gtt atc atg ata ctg aag cgc agc acc agg 12004Val Glu Phe Met Asp Thr Val Ile Met Ile Leu Lys Arg Ser Thr Arg 140 145 150 caa ata agc ttc ctc cac gtt tat cat cat tct tca att tcc ctc att 12052Gln Ile Ser Phe Leu His Val Tyr His His Ser Ser Ile Ser Leu Ile 155 160 165 170 tgg tgg gct att gct cat cac gct cct ggc ggt gaa gca tat tgg tct 12100Trp Trp Ala Ile Ala His His Ala Pro Gly Gly Glu Ala Tyr Trp Ser 175 180 185 gcg gct ctg aac tca gga gtg cat gtt ctc atg tat gcg tat tac ttc 12148Ala Ala Leu Asn Ser Gly Val His Val Leu Met Tyr Ala Tyr Tyr Phe 190 195 200 ttg gct gcc tgc ctt cga agt agc cca aag tta aaa aat aag tac ctt 12196Leu Ala Ala Cys Leu Arg Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu 205 210 215 ttt tgg ggc agg tac ttg aca caa ttc caa atg ttc cag ttt atg ctg 12244Phe Trp Gly Arg Tyr Leu Thr Gln Phe Gln Met Phe Gln Phe Met Leu 220 225 230 aac tta gtg cag gct tac tac gac atg aaa acg aat gcg cca tat cca 12292Asn Leu Val Gln Ala Tyr Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro 235 240 245 250 caa tgg ctg atc aag att ttg ttc tac tac atg atc tcg ttg ctg ttt 12340Gln Trp Leu Ile Lys Ile Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe 255 260 265 ctt ttc ggc aat ttt tac gta caa aaa tac atc aaa ccc tct gac gga 12388Leu Phe Gly Asn Phe Tyr Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly 270 275 280 aag caa aag gga gct aaa act gag tga tctagaaggc ctcctgcttt 12435Lys Gln Lys Gly Ala Lys Thr Glu 285 290 aatgagatat gcgagacgcc tatgatcgca tgatatttgc tttcaattct gttgtgcacg 12495ttgtaaaaaa cctgagcatg tgtagctcag atccttaccg ccggtttcgg ttcattctaa

12555tgaatatatc acccgttact atcgtatttt tatgaataat attctccgtt caatttactg 12615attgtccgtc gagcaaattt acacattgcc actaaacgtc taaacccttg taatttgttt 12675ttgttttact atgtgtgtta tgtatttgat ttgcgataaa tttttatatt tggtactaaa 12735tttataacac cttttatgct aacgtttgcc aacacttagc aatttgcaag ttgattaatt 12795gattctaaat tatttttgtc ttctaaatac atatactaat caactggaaa tgtaaatatt 12855tgctaatatt tctactatag gagaattaaa gtgagtgaat atggtaccac aaggtttgga 12915gatttaattg ttgcaatgct gcatggatgg catatacacc aaacattcaa taattcttga 12975ggataataat ggtaccacac aagatttgag gtgcatgaac gtcacgtgga caaaaggttt 13035agtaattttt caagacaaca atgttaccac acacaagttt tgaggtgcat gcatggatgc 13095cctgtggaaa gtttaaaaat attttggaaa tgatttgcat ggaagccatg tgtaaaacca 13155tgacatccac ttggaggatg caataatgaa gaaaactaca aatttacatg caactagtta 13215tgcatgtagt ctatataatg aggattttgc aatactttca ttcatacaca ctcactaagt 13275tttacacgat tataatttct tcatagccag cggatcc atg gta ttc gcg ggc ggt 13330 Met Val Phe Ala Gly Gly 295 gga ctt cag cag ggc tct ctc gaa gaa aac atc gac gtc gag cac att 13378Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn Ile Asp Val Glu His Ile 300 305 310 gcc agt atg tct ctc ttc agc gac ttc ttc agt tat gtg tct tca act 13426Ala Ser Met Ser Leu Phe Ser Asp Phe Phe Ser Tyr Val Ser Ser Thr 315 320 325 gtt ggt tcg tgg agc gta cac agt ata caa cct ttg aag cgc ctg acg 13474Val Gly Ser Trp Ser Val His Ser Ile Gln Pro Leu Lys Arg Leu Thr 330 335 340 agt aag aag cgt gtt tcg gaa agc gct gcc gtg caa tgt ata tca gct 13522Ser Lys Lys Arg Val Ser Glu Ser Ala Ala Val Gln Cys Ile Ser Ala 345 350 355 360 gaa gtt cag aga aat tcg agt acc cag gga act gcg gag gca ctc gca 13570Glu Val Gln Arg Asn Ser Ser Thr Gln Gly Thr Ala Glu Ala Leu Ala 365 370 375 gaa tca gtc gtg aag ccc acg aga cga agg tca tct cag tgg aag aag 13618Glu Ser Val Val Lys Pro Thr Arg Arg Arg Ser Ser Gln Trp Lys Lys 380 385 390 tcg aca cac ccc cta tca gaa gta gca gta cac aac aag cca agc gat 13666Ser Thr His Pro Leu Ser Glu Val Ala Val His Asn Lys Pro Ser Asp 395 400 405 tgc tgg att gtt gta aaa aac aag gtg tat gat gtt tcc aat ttt gcg 13714Cys Trp Ile Val Val Lys Asn Lys Val Tyr Asp Val Ser Asn Phe Ala 410 415 420 gac gag cat ccc gga gga tca gtt att agt act tat ttt gga cga gac 13762Asp Glu His Pro Gly Gly Ser Val Ile Ser Thr Tyr Phe Gly Arg Asp 425 430 435 440 ggc aca gat gtt ttc tct agt ttt cat gca gct tct aca tgg aaa att 13810Gly Thr Asp Val Phe Ser Ser Phe His Ala Ala Ser Thr Trp Lys Ile 445 450 455 ctt caa gac ttt tac att ggt gac gtg gag agg gtg gag ccg act cca 13858Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu Arg Val Glu Pro Thr Pro 460 465 470 gag ctg ctg aaa gat ttc cga gaa atg aga gct ctt ttc ctg agg gag 13906Glu Leu Leu Lys Asp Phe Arg Glu Met Arg Ala Leu Phe Leu Arg Glu 475 480 485 caa ctt ttc aaa agt tcg aaa ttg tac tat gtt atg aag ctg ctc acg 13954Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr Val Met Lys Leu Leu Thr 490 495 500 aat gtt gct att ttt gct gcg agc att gca ata ata tgt tgg agc aag 14002Asn Val Ala Ile Phe Ala Ala Ser Ile Ala Ile Ile Cys Trp Ser Lys 505 510 515 520 act att tca gcg gtt ttg gct tca gct tgt atg atg gct ctg tgt ttc 14050Thr Ile Ser Ala Val Leu Ala Ser Ala Cys Met Met Ala Leu Cys Phe 525 530 535 caa cag tgc gga tgg cta tcc cat gat ttt ctc cac aat cag gtg ttt 14098Gln Gln Cys Gly Trp Leu Ser His Asp Phe Leu His Asn Gln Val Phe 540 545 550 gag aca cgc tgg ctt aat gaa gtt gtc ggg tat gtg atc ggc aac gcc 14146Glu Thr Arg Trp Leu Asn Glu Val Val Gly Tyr Val Ile Gly Asn Ala 555 560 565 gtt ctg ggg ttt agt aca ggg tgg tgg aag gag aag cat aac ctt cat 14194Val Leu Gly Phe Ser Thr Gly Trp Trp Lys Glu Lys His Asn Leu His 570 575 580 cat gct gct cca aat gaa tgc gat cag act tac caa cca att gat gaa 14242His Ala Ala Pro Asn Glu Cys Asp Gln Thr Tyr Gln Pro Ile Asp Glu 585 590 595 600 gat att gat act ctc ccc ctc att gcc tgg agc aag gac ata ctg gcc 14290Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp Ser Lys Asp Ile Leu Ala 605 610 615 aca gtt gag aat aag aca ttc ttg cga atc ctc caa tac cag cat ctg 14338Thr Val Glu Asn Lys Thr Phe Leu Arg Ile Leu Gln Tyr Gln His Leu 620 625 630 ttc ttc atg ggt ctg tta ttt ttc gcc cgt ggt agt tgg ctc ttt tgg 14386Phe Phe Met Gly Leu Leu Phe Phe Ala Arg Gly Ser Trp Leu Phe Trp 635 640 645 agc tgg aga tat acc tct aca gca gtg ctc tca cct gtc gac agg ttg 14434Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu Ser Pro Val Asp Arg Leu 650 655 660 ttg gag aag gga act gtt ctg ttt cac tac ttt tgg ttc gtc ggg aca 14482Leu Glu Lys Gly Thr Val Leu Phe His Tyr Phe Trp Phe Val Gly Thr 665 670 675 680 gcg tgc tat ctt ctc cct ggt tgg aag cca tta gta tgg atg gcg gtg 14530Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro Leu Val Trp Met Ala Val 685 690 695 act gag ctc atg tcc ggc atg ctg ctg ggc ttt gta ttt gta ctt agc 14578Thr Glu Leu Met Ser Gly Met Leu Leu Gly Phe Val Phe Val Leu Ser 700 705 710 cac aat ggg atg gag gtt tat aat tcg tct aaa gaa ttc gtg agt gca 14626His Asn Gly Met Glu Val Tyr Asn Ser Ser Lys Glu Phe Val Ser Ala 715 720 725 cag atc gta tcc aca cgg gat atc aaa gga aac ata ttc aac gac tgg 14674Gln Ile Val Ser Thr Arg Asp Ile Lys Gly Asn Ile Phe Asn Asp Trp 730 735 740 ttc act ggt ggc ctt aac agg caa ata gag cat cat ctt ttc cca aca 14722Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu His His Leu Phe Pro Thr 745 750 755 760 atg ccc agg cat aat tta aac aaa ata gca cct aga gtg gag gtg ttc 14770Met Pro Arg His Asn Leu Asn Lys Ile Ala Pro Arg Val Glu Val Phe 765 770 775 tgt aag aaa cac ggt ctg gtg tac gaa gac gta tct att gct acc ggc 14818Cys Lys Lys His Gly Leu Val Tyr Glu Asp Val Ser Ile Ala Thr Gly 780 785 790 act tgc aag gtt ttg aaa gca ttg aag gaa gtc gcg gag gct gcg gca 14866Thr Cys Lys Val Leu Lys Ala Leu Lys Glu Val Ala Glu Ala Ala Ala 795 800 805 gag cag cat gct acc acc agt taa gctagcgtta accctgcttt aatgagatat 14920Glu Gln His Ala Thr Thr Ser 810 815 gcgagacgcc tatgatcgca tgatatttgc tttcaattct gttgtgcacg ttgtaaaaaa 14980cctgagcatg tgtagctcag atccttaccg ccggtttcgg ttcattctaa tgaatatatc 15040acccgttact atcgtatttt tatgaataat attctccgtt caatttactg attgtccgtc 15100gagcaaattt acacattgcc actaaacgtc taaacccttg taatttgttt ttgttttact 15160atgtgtgtta tgtatttgat ttgcgataaa tttttatatt tggtactaaa tttataacac 15220cttttatgct aacgtttgcc aacacttagc aatttgcaag ttgattaatt gattctaaat 15280tatttttgtc ttctaaatac atatactaat caactggaaa tgtaaatatt tgctaatatt 15340tctactatag gagaattaaa gtgagtgaat atggtaccac aaggtttgga gatttaattg 15400ttgcaatgct gcatggatgg catatacacc aaacattcaa taattcttga ggataataat 15460ggtaccacac aagatttgag gtgcatgaac gtcacgtgga caaaaggttt agtaattttt 15520caagacaaca atgttaccac acacaagttt tgaggtgcat gcatggatgc cctgtggaaa 15580gtttaaaaat attttggaaa tgatttgcat ggaagccatg tgtaaaacca tgacatccac 15640ttggaggatg caataatgaa gaaaactaca aatttacatg caactagtta tgcatgtagt 15700ctatataatg aggattttgc aatactttca ttcatacaca ctcactaagt tttacacgat 15760tataatttct tcatagccag cagatctaaa atg gct ccg gat gcg gat aag ctt 15814 Met Ala Pro Asp Ala Asp Lys Leu 820 cga caa cgc cag acg act gcg gta gcg aag cac aat gct gct acc ata 15862Arg Gln Arg Gln Thr Thr Ala Val Ala Lys His Asn Ala Ala Thr Ile 825 830 835 tcg acg cag gaa cgc ctt tgc agt ctg tct tcg ctc aaa ggc gaa gaa 15910Ser Thr Gln Glu Arg Leu Cys Ser Leu Ser Ser Leu Lys Gly Glu Glu 840 845 850 855 gtc tgc atc gac gga atc atc tat gac ctc caa tca ttc gat cat ccc 15958Val Cys Ile Asp Gly Ile Ile Tyr Asp Leu Gln Ser Phe Asp His Pro 860 865 870 ggg ggt gaa acg atc aaa atg ttt ggt ggc aac gat gtc act gta cag 16006Gly Gly Glu Thr Ile Lys Met Phe Gly Gly Asn Asp Val Thr Val Gln 875 880 885 tac aag atg att cac ccg tac cat acc gag aag cat ttg gaa aag atg 16054Tyr Lys Met Ile His Pro Tyr His Thr Glu Lys His Leu Glu Lys Met 890 895 900 aag cgt gtc ggc aag gtg acg gat ttc gtc tgc gag tac aag ttc gat 16102Lys Arg Val Gly Lys Val Thr Asp Phe Val Cys Glu Tyr Lys Phe Asp 905 910 915 acc gaa ttt gaa cgc gaa atc aaa cga gaa gtc ttc aag att gtg cga 16150Thr Glu Phe Glu Arg Glu Ile Lys Arg Glu Val Phe Lys Ile Val Arg 920 925 930 935 cga ggc aag gat ttc ggt act ttg gga tgg ttc ttc cgt gcg ttt tgc 16198Arg Gly Lys Asp Phe Gly Thr Leu Gly Trp Phe Phe Arg Ala Phe Cys 940 945 950 tac att gcc att ttc ttc tac ctg cag tac cat tgg gtc acc acg gga 16246Tyr Ile Ala Ile Phe Phe Tyr Leu Gln Tyr His Trp Val Thr Thr Gly 955 960 965 acc tct tgg ctg ctg gcc gtg gcc tac gga atc tcc caa gcg atg att 16294Thr Ser Trp Leu Leu Ala Val Ala Tyr Gly Ile Ser Gln Ala Met Ile 970 975 980 ggc atg aat gtc cag cac gat gcc aac cac ggg gcc acc tcc aag cgt 16342Gly Met Asn Val Gln His Asp Ala Asn His Gly Ala Thr Ser Lys Arg 985 990 995 ccc tgg gtc aac gac atg cta ggc ctc ggt gcg gat ttt att ggt 16387Pro Trp Val Asn Asp Met Leu Gly Leu Gly Ala Asp Phe Ile Gly 1000 1005 1010 ggt tcc aag tgg ctc tgg cag gaa caa cac tgg acc cac cac gct 16432Gly Ser Lys Trp Leu Trp Gln Glu Gln His Trp Thr His His Ala 1015 1020 1025 tac acc aat cac gcc gag atg gat ccc gat agc ttt ggt gcc gaa 16477Tyr Thr Asn His Ala Glu Met Asp Pro Asp Ser Phe Gly Ala Glu 1030 1035 1040 cca atg ctc cta ttc aac gac tat ccc ttg gat cat ccc gct cgt 16522Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp His Pro Ala Arg 1045 1050 1055 acc tgg cta cat cgc ttt caa gca ttc ttt tac atg ccc gtc ttg 16567Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met Pro Val Leu 1060 1065 1070 gct gga tac tgg ttg tcc gct gtc ttc aat cca caa att ctt gac 16612Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile Leu Asp 1075 1080 1085 ctc cag caa cgc ggc gca ctt tcc gtc ggt atc cgt ctc gac aac 16657Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp Asn 1090 1095 1100 gct ttc att cac tcg cga cgc aag tat gcg gtt ttc tgg cgg gct 16702Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 1105 1110 1115 gtg tac att gcg gtg aac gtg att gct ccg ttt tac aca aac tcc 16747Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser 1120 1125 1130 ggc ctc gaa tgg tcc tgg cgt gtc ttt gga aac atc atg ctc atg 16792Gly Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met 1135 1140 1145 ggt gtg gcg gaa tcg ctc gcg ctg gcg gtc ctg ttt tcg ttg tcg 16837Gly Val Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser 1150 1155 1160 cac aat ttc gaa tcc gcg gat cgc gat ccg acc gcc cca ctg aaa 16882His Asn Phe Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys 1165 1170 1175 aag acg gga gaa cca gtc gac tgg ttc aag aca cag gtc gaa act 16927Lys Thr Gly Glu Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr 1180 1185 1190 tcc tgc act tac ggt gga ttc ctt tcc ggt tgc ttc acg gga ggt 16972Ser Cys Thr Tyr Gly Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly 1195 1200 1205 ctc aac ttt cag gtt gaa cac cac ttg ttc cca cgc atg agc agc 17017Leu Asn Phe Gln Val Glu His His Leu Phe Pro Arg Met Ser Ser 1210 1215 1220 gct tgg tat ccc tac att gcc ccc aag gtc cgc gaa att tgc gcc 17062Ala Trp Tyr Pro Tyr Ile Ala Pro Lys Val Arg Glu Ile Cys Ala 1225 1230 1235 aaa cac ggc gtc cac tac gcc tac tac ccg tgg atc cac caa aac 17107Lys His Gly Val His Tyr Ala Tyr Tyr Pro Trp Ile His Gln Asn 1240 1245 1250 ttt ctc tcc acc gtc cgc tac atg cac gcg gcc ggg acc ggt gcc 17152Phe Leu Ser Thr Val Arg Tyr Met His Ala Ala Gly Thr Gly Ala 1255 1260 1265 aac tgg cgc cag atg gcc aga gaa aat ccc ttg acc gga cgg gcg 17197Asn Trp Arg Gln Met Ala Arg Glu Asn Pro Leu Thr Gly Arg Ala 1270 1275 1280 taa agatctgccg gcatcgatcc cgggccatgg cctgctttaa tgagatatgc 17250gagacgccta tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc 17310tgagcatgtg tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac 17370ccgttactat cgtattttta tgaataatat tctccgttca atttactgat tgtccgtcga 17430cgagctcggc gcgcctctag aggatcgatg aattcagatc ggctgagtgg ctccttcaac 17490gttgcggttc tgtcagttcc aaacgtaaaa cggcttgtcc cgcgtcatcg gcgggggtca 17550taacgtgact cccttaattc tccgctcatg atcagattgt cgtttcccgc cttcagttta 17610aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt 17670agaataatcg gatatttaaa agggcgtgaa aaggtttatc cttcgtccat ttgtatgtgc 17730atgccaacca cagggttccc ca 1775220290PRTArtificial sequenceDelta-6-elongase of Physcomitrella patens 20Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser 1 5 10 15 Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp 20 25 30 Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 40 45 Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 50 55 60 Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155 160 Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165 170

175 His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190 Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200 205 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210 215 220 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr 225 230 235 240 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile 245 250 255 Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr 260 265 270 Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 275 280 285 Thr Glu 290 21525PRTArtificial sequenceDelta-6-desaturase of Physcomitrella patens 21Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn 1 5 10 15 Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe 20 25 30 Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln 35 40 45 Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala 50 55 60 Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly 65 70 75 80 Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg 85 90 95 Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val 100 105 110 His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr 115 120 125 Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser 130 135 140 Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala 145 150 155 160 Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu 165 170 175 Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe Arg Glu Met Arg 180 185 190 Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr 195 200 205 Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe Ala Ala Ser Ile Ala 210 215 220 Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val Leu Ala Ser Ala Cys 225 230 235 240 Met Met Ala Leu Cys Phe Gln Gln Cys Gly Trp Leu Ser His Asp Phe 245 250 255 Leu His Asn Gln Val Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly 260 265 270 Tyr Val Ile Gly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys 275 280 285 Glu Lys His Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr 290 295 300 Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp 305 310 315 320 Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile 325 330 335 Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg 340 345 350 Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu 355 360 365 Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr 370 375 380 Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro 385 390 395 400 Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met Leu Leu Gly 405 410 415 Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val Tyr Asn Ser Ser 420 425 430 Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr Arg Asp Ile Lys Gly 435 440 445 Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu 450 455 460 His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Lys Ile Ala 465 470 475 480 Pro Arg Val Glu Val Phe Cys Lys Lys His Gly Leu Val Tyr Glu Asp 485 490 495 Val Ser Ile Ala Thr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu 500 505 510 Val Ala Glu Ala Ala Ala Glu Gln His Ala Thr Thr Ser 515 520 525 22469PRTArtificial sequenceDelta-5-desaturase of Phaeodactylum tricornutum 22Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val 1 5 10 15 Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser 20 25 30 Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr 35 40 45 Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe 50 55 60 Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His 65 70 75 80 Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp 85 90 95 Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys 100 105 110 Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu 115 120 125 Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu 130 135 140 Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala 145 150 155 160 Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala 165 170 175 Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly 180 185 190 Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln 195 200 205 His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp 210 215 220 Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp 225 230 235 240 His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met 245 250 255 Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile 260 265 270 Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp 275 280 285 Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala 290 295 300 Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly 305 310 315 320 Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val 325 330 335 Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe 340 345 350 Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu 355 360 365 Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly 370 375 380 Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu 385 390 395 400 His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala 405 410 415 Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr 420 425 430 Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His 435 440 445 Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro 450 455 460 Leu Thr Gly Arg Ala 465 2326DNAArtificial sequencepolylinker sequence 23gaattcggcg cgccgagctc ctcgag 2624265DNAArtificial sequencepolylinker-terminator-polylinkers sequence 24ccaccgcggt gggcggccgc ctgcagtcta gaaggcctcc tgctttaatg agatatgcga 60gacgcctatg atcgcatgat atttgctttc aattctgttg tgcacgttgt aaaaaacctg 120agcatgtgta gctcagatcc ttaccgccgg tttcggttca ttctaatgaa tatatcaccc 180gttactatcg tatttttatg aataatattc tccgttcaat ttactgattg tccgtcgacg 240aattcgagct cggcgcgcca agctt 26525257DNAArtificial sequencepolylinker-terminator-polylinkers sequence 25ggatccgata tcgggcccgc tagcgttaac cctgctttaa tgagatatgc gagacgccta 60tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg 120tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac ccgttactat 180cgtattttta tgaataatat tctccgttca atttactgat tgtccgtcga cgaattcgag 240ctcggcgcgc caagctt 257265410DNAArtificial sequenceplant expression vector with one promoter-terminator expression cassette 26ttttggaaat gatttgcatg gaagccatgt gtaaaaccat gacatccact tggaggatgc 60aataatgaag aaaactacaa atttacatgc aactagttat gcatgtagtc tatataatga 120ggattttgca atactttcat tcatacacac tcactaagtt ttacacgatt ataatttctt 180catagccagc ggatccgata tcgggcccgc tagcgttaac cctgctttaa tgagatatgc 240gagacgccta tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc 300tgagcatgtg tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac 360ccgttactat cgtattttta tgaataatat tctccgttca atttactgat tgtccgtcga 420gcaaatttac acattgccac taaacgtcta aacccttgta atttgttttt gttttactat 480gtgtgttatg tatttgattt gcgataaatt tttatatttg gtactaaatt tataacacct 540tttatgctaa cgtttgccaa cacttagcaa tttgcaagtt gattaattga ttctaaatta 600tttttgtctt ctaaatacat atactaatca actggaaatg taaatatttg ctaatatttc 660tactatagga gaattaaagt gagtgaatat ggtaccacaa ggtttggaga tttaattgtt 720gcaatgctgc atggatggca tatacaccaa acattcaata attcttgagg ataataatgg 780taccacacaa gatttgaggt gcatgaacgt cacgtggaca aaaggtttag taatttttca 840agacaacaat gttaccacac acaagttttg aggtgcatgc atggatgccc tgtggaaagt 900ttaaaaatat tttggaaatg atttgcatgg aagccatgtg taaaaccatg acatccactt 960ggaggatgca ataatgaaga aaactacaaa tttacatgca actagttatg catgtagtct 1020atataatgag gattttgcaa tactttcatt catacacact cactaagttt tacacgatta 1080taatttcttc atagccagca gatctgccgg catcgatccc gggccatggc ctgctttaat 1140gagatatgcg agacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg 1200taaaaaacct gagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga 1260atatatcacc cgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt 1320gtccgtcgac gagctcggcg cgccaagctt ggcgtaatca tggtcatagc tgtttcctgt 1380gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 1440agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 1500tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 1560aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 1620cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 1680atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 1740taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1800aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1860tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1920gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1980cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 2040cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 2100atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 2160tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 2220ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 2280acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 2340aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 2400aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 2460tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 2520cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 2580catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 2640ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 2700aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 2760ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2820caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2880attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2940agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 3000actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 3060ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 3120ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 3180gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 3240atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 3300cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 3360gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 3420gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 3480ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 3540gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 3600tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 3660ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 3720ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 3780aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct 3840gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 3900agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 3960ttgtaaaacg acggccagtg aattcggcgc gccgagctcc tcgagcaaat ttacacattg 4020ccactaaacg tctaaaccct tgtaatttgt ttttgtttta ctatgtgtgt tatgtatttg 4080atttgcgata aatttttata tttggtacta aatttataac accttttatg ctaacgtttg 4140ccaacactta gcaatttgca agttgattaa ttgattctaa attatttttg tcttctaaat 4200acatatacta atcaactgga aatgtaaata tttgctaata tttctactat aggagaatta 4260aagtgagtga atatggtacc acaaggtttg gagatttaat tgttgcaatg ctgcatggat 4320ggcatataca ccaaacattc aataattctt gaggataata atggtaccac acaagatttg 4380aggtgcatga acgtcacgtg gacaaaaggt ttagtaattt ttcaagacaa caatgttacc 4440acacacaagt tttgaggtgc atgcatggat gccctgtgga aagtttaaaa atattttgga 4500aatgatttgc atggaagcca tgtgtaaaac catgacatcc acttggagga tgcaataatg 4560aagaaaacta caaatttaca tgcaactagt tatgcatgta gtctatataa tgaggatttt 4620gcaatacttt cattcataca cactcactaa gttttacacg attataattt cttcatagcc 4680agcccaccgc ggtgggcggc cgcctgcagt ctagaaggcc tcctgcttta atgagatatg 4740cgagacgcct atgatcgcat gatatttgct ttcaattctg ttgtgcacgt tgtaaaaaac 4800ctgagcatgt gtagctcaga tccttaccgc cggtttcggt tcattctaat gaatatatca 4860cccgttacta tcgtattttt atgaataata ttctccgttc aatttactga ttgtccgtcg 4920agcaaattta cacattgcca ctaaacgtct aaacccttgt aatttgtttt tgttttacta 4980tgtgtgttat gtatttgatt tgcgataaat ttttatattt ggtactaaat ttataacacc 5040ttttatgcta acgtttgcca acacttagca atttgcaagt tgattaattg attctaaatt 5100atttttgtct tctaaataca tatactaatc aactggaaat gtaaatattt gctaatattt 5160ctactatagg agaattaaag tgagtgaata tggtaccaca aggtttggag atttaattgt 5220tgcaatgctg catggatggc atatacacca aacattcaat aattcttgag gataataatg 5280gtaccacaca agatttgagg tgcatgaacg tcacgtggac aaaaggttta gtaatttttc 5340aagacaacaa tgttaccaca cacaagtttt gaggtgcatg catggatgcc ctgtggaaag 5400tttaaaaata 54102712093DNAArtificial sequenceplant expression vector with one promoter-terminator expression cassette 27gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc

tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagccca ccgcggtggg cggccgcctg cagtctagaa ggcctcctgc tttaatgaga 11580tatgcgagac gcctatgatc gcatgatatt tgctttcaat tctgttgtgc acgttgtaaa 11640aaacctgagc atgtgtagct cagatcctta ccgccggttt cggttcattc taatgaatat 11700atcacccgtt actatcgtat ttttatgaat aatattctcc gttcaattta ctgattgtcc 11760gtcgacgaat tcgagctcgg cgcgcctcta gaggatcgat gaattcagat cggctgagtg 11820gctccttcaa cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc 11880ggcgggggtc ataacgtgac tcccttaatt ctccgctcat gatcagattg tcgtttcccg 11940ccttcagttt aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa 12000gagcgtttat tagaataatc ggatatttaa aagggcgtga aaaggtttat ccttcgtcca 12060tttgtatgtg catgccaacc acagggttcc cca 120932812085DNAArtificial sequenceplant expression vector with one promoter-terminator expression cassette 28gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc

cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagcgga tccgatatcg ggcccgctag cgttaaccct gctttaatga gatatgcgag 11580acgcctatga tcgcatgata tttgctttca attctgttgt gcacgttgta aaaaacctga 11640gcatgtgtag ctcagatcct taccgccggt ttcggttcat tctaatgaat atatcacccg 11700ttactatcgt atttttatga ataatattct ccgttcaatt tactgattgt ccgtcgacga 11760attcgagctc ggcgcgcctc tagaggatcg atgaattcag atcggctgag tggctccttc 11820aacgttgcgg ttctgtcagt tccaaacgta aaacggcttg tcccgcgtca tcggcggggg 11880tcataacgtg actcccttaa ttctccgctc atgatcagat tgtcgtttcc cgccttcagt 11940ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 12000attagaataa tcggatattt aaaagggcgt gaaaaggttt atccttcgtc catttgtatg 12060tgcatgccaa ccacagggtt cccca 120852912079DNAArtificial sequenceplant expression vector with one promoter-terminator expression cassette 29gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac

gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagcaga tctgccggca tcgatcccgg gccatggcct gctttaatga gatatgcgag 11580acgcctatga tcgcatgata tttgctttca attctgttgt gcacgttgta aaaaacctga 11640gcatgtgtag ctcagatcct taccgccggt ttcggttcat tctaatgaat atatcacccg 11700ttactatcgt atttttatga ataatattct ccgttcaatt tactgattgt ccgtcgacga 11760gctcggcgcg cctctagagg atcgatgaat tcagatcggc tgagtggctc cttcaacgtt 11820gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg ggggtcataa 11880cgtgactccc ttaattctcc gctcatgatc agattgtcgt ttcccgcctt cagtttaaac 11940tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga 12000ataatcggat atttaaaagg gcgtgaaaag gtttatcctt cgtccatttg tatgtgcatg 12060ccaaccacag ggttcccca 120793013002DNAArtificial sequenceplant expression vector with two promoter-terminator expression cassettes 30gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat

catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagccca ccgcggtggg cggccgcctg cagtctagaa ggcctcctgc tttaatgaga 11580tatgcgagac gcctatgatc gcatgatatt tgctttcaat tctgttgtgc acgttgtaaa 11640aaacctgagc atgtgtagct cagatcctta ccgccggttt cggttcattc taatgaatat 11700atcacccgtt actatcgtat ttttatgaat aatattctcc gttcaattta ctgattgtcc 11760gtcgagcaaa tttacacatt gccactaaac gtctaaaccc ttgtaatttg tttttgtttt 11820actatgtgtg ttatgtattt gatttgcgat aaatttttat atttggtact aaatttataa 11880caccttttat gctaacgttt gccaacactt agcaatttgc aagttgatta attgattcta 11940aattattttt gtcttctaaa tacatatact aatcaactgg aaatgtaaat atttgctaat 12000atttctacta taggagaatt aaagtgagtg aatatggtac cacaaggttt ggagatttaa 12060ttgttgcaat gctgcatgga tggcatatac accaaacatt caataattct tgaggataat 12120aatggtacca cacaagattt gaggtgcatg aacgtcacgt ggacaaaagg tttagtaatt 12180tttcaagaca acaatgttac cacacacaag ttttgaggtg catgcatgga tgccctgtgg 12240aaagtttaaa aatattttgg aaatgatttg catggaagcc atgtgtaaaa ccatgacatc 12300cacttggagg atgcaataat gaagaaaact acaaatttac atgcaactag ttatgcatgt 12360agtctatata atgaggattt tgcaatactt tcattcatac acactcacta agttttacac 12420gattataatt tcttcatagc cagcggatcc gatatcgggc ccgctagcgt taaccctgct 12480ttaatgagat atgcgagacg cctatgatcg catgatattt gctttcaatt ctgttgtgca 12540cgttgtaaaa aacctgagca tgtgtagctc agatccttac cgccggtttc ggttcattct 12600aatgaatata tcacccgtta ctatcgtatt tttatgaata atattctccg ttcaatttac 12660tgattgtccg tcgacgaatt cgagctcggc gcgcctctag aggatcgatg aattcagatc 12720ggctgagtgg ctccttcaac gttgcggttc tgtcagttcc aaacgtaaaa cggcttgtcc 12780cgcgtcatcg gcgggggtca taacgtgact cccttaattc tccgctcatg atcagattgt 12840cgtttcccgc cttcagttta aactatcagt gtttgacagg atatattggc gggtaaacct 12900aagagaaaag agcgtttatt agaataatcg gatatttaaa agggcgtgaa aaggtttatc 12960cttcgtccat ttgtatgtgc atgccaacca cagggttccc ca 130023113905DNAArtificial sequenceplant expression vector with three promoter-terminator expression cassettes 31gatctggcgc cggccagcga gacgagcaag attggccgcc gcccgaaacg atccgacagc 60gcgcccagca caggtgcgca ggcaaattgc accaacgcat acagcgccag cagaatgcca 120tagtgggcgg tgacgtcgtt cgagtgaacc agatcgcgca ggaggcccgg cagcaccggc 180ataatcaggc cgatgccgac agcgtcgagc gcgacagtgc tcagaattac gatcaggggt 240atgttgggtt tcacgtctgg cctccggacc agcctccgct ggtccgattg aacgcgcgga 300ttctttatca ctgataagtt ggtggacata ttatgtttat cagtgataaa gtgtcaagca 360tgacaaagtt gcagccgaat acagtgatcc gtgccgccct ggacctgttg aacgaggtcg 420gcgtagacgg tctgacgaca cgcaaactgg cggaacggtt gggggttcag cagccggcgc 480tttactggca cttcaggaac aagcgggcgc tgctcgacgc actggccgaa gccatgctgg 540cggagaatca tacgcattcg gtgccgagag ccgacgacga ctggcgctca tttctgatcg 600ggaatgcccg cagcttcagg caggcgctgc tcgcctaccg cgatggcgcg cgcatccatg 660ccggcacgcg accgggcgca ccgcagatgg aaacggccga cgcgcagctt cgcttcctct 720gcgaggcggg tttttcggcc ggggacgccg tcaatgcgct gatgacaatc agctacttca 780ctgttggggc cgtgcttgag gagcaggccg gcgacagcga tgccggcgag cgcggcggca 840ccgttgaaca ggctccgctc tcgccgctgt tgcgggccgc gatagacgcc ttcgacgaag 900ccggtccgga cgcagcgttc gagcagggac tcgcggtgat tgtcgatgga ttggcgaaaa 960ggaggctcgt tgtcaggaac gttgaaggac cgagaaaggg tgacgattga tcaggaccgc 1020tgccggagcg caacccactc actacagcag agccatgtag acaacatccc ctcccccttt 1080ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt 1140ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctctt ccgcttcctc 1200gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 1260ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 1320aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 1380ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 1440aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 1500gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttt 1560ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt 1620tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac 1680ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca 1740ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg 1800ctaccgccgg cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga 1860agggcagccc acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa 1920aggcggcggc ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca 1980aaatcacggg cgtcgtggac tatgagcacg tccgcgagct ggcccgcatc aatggcgacc 2040tgggccgcct gggcggcctg ctgaaactct ggctcaccga cgacccgcgc acggcgcggt 2100tcggtgatgc cacgatcctc gccctgctgg cgaagatcga agagaagcag gacgagcttg 2160gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc atgacttttt tagccgctaa 2220aacggccggg gggtgcgcgt gattgccaag cacgtcccca tgcgctccat caagaagagc 2280gacttcgcgg agctggtgaa gtacatcacc gacgagcaag gcaagaccga gcgcctttgc 2340gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg gccctgcaaa 2400cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc gttgtggata 2460cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc 2520cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg 2580gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat 2640gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac 2700tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca gatgaggggc 2760gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt 2820ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat 2880aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc cgaagggggg 2940tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc ccccaggggc 3000tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agcgctggca gtccttgcca 3060ttgccgggat cggggcagta acgggatggg cgatcagccc gagcgcgacg cccggaagca 3120ttgacgtgcc gcaggtgctg gcatcgacat tcagcgacca ggtgccgggc agtgagggcg 3180gcggcctggg tggcggcctg cccttcactt cggccgtcgg ggcattcacg gacttcatgg 3240cggggccggc aatttttacc ttgggcattc ttggcatagt ggtcgcgggt gccgtgctcg 3300tgttcggggg tgcgataaac ccagcgaacc atttgaggtg ataggtaaga ttataccgag 3360gtatgaaaac gagaattgga cctttacaga attactctat gaagcgccat atttaaaaag 3420ctaccaagac gaagaggatg aagaggatga ggaggcagat tgccttgaat atattgacaa 3480tactgataag ataatatatc ttttatatag aagatatcgc cgtatgtaag gatttcaggg 3540ggcaaggcat aggcagcgcg cttatcaata tatctataga atgggcaaag cataaaaact 3600tgcatggact aatgcttgaa acccaggaca ataaccttat agcttgtaaa ttctatcata 3660attgggtaat gactccaact tattgatagt gttttatgtt cagataatgc ccgatgactt 3720tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc cgtgccaggt 3780gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg ctgattacgt 3840gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc catatcacca 3900cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg cgttcaccga 3960atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc cagcgctggc 4020gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg atgacgtcac 4080tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt gacgtaaaat 4140cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc cattcatggc 4200catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga actgcagttg 4260ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc ttttgccgtt 4320acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagacacag aagccactgg 4380agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca cccataattg 4440tggtttcaaa atcggctccg tcgatactat gttatacgcc aactttgaaa acaactttga 4500aaaagctgtt ttctggtatt taaggtttta gaatgcaagg aacagtgaat tggagttcgt 4560cttgttataa ttagcttctt ggggtatctt taaatactgt agaaaagagg aaggaaataa 4620taaatggcta aaatgagaat atcaccggaa ttgaaaaaac tgatcgaaaa ataccgctgc 4680gtaaaagata cggaaggaat gtctcctgct aaggtatata agctggtggg agaaaatgaa 4740aacctatatt taaaaatgac ggacagccgg tataaaggga ccacctatga tgtggaacgg 4800gaaaaggaca tgatgctatg gctggaagga aagctgcctg ttccaaaggt cctgcacttt 4860gaacggcatg atggctggag caatctgctc atgagtgagg ccgatggcgt cctttgctcg 4920gaagagtatg aagatgaaca aagccctgaa aagattatcg agctgtatgc ggagtgcatc 4980aggctctttc actccatcga catatcggat tgtccctata cgaatagctt agacagccgc 5040ttagccgaat tggattactt actgaataac gatctggccg atgtggattg cgaaaactgg 5100gaagaagaca ctccatttaa agatccgcgc gagctgtatg attttttaaa gacggaaaag 5160cccgaagagg aacttgtctt ttcccacggc gacctgggag acagcaacat ctttgtgaaa 5220gatggcaaag taagtggctt tattgatctt gggagaagcg gcagggcgga caagtggtat 5280gacattgcct tctgcgtccg gtcgatcagg gaggatatcg gggaagaaca gtatgtcgag 5340ctattttttg acttactggg gatcaagcct gattgggaga aaataaaata ttatatttta 5400ctggatgaat tgttttagta cctagatgtg gcgcaacgat gccggcgaca agcaggagcg 5460caccgacttc ttccgcatca agtgttttgg ctctcaggcc gaggcccacg gcaagtattt 5520gggcaagggg tcgctggtat tcgtgcaggg caagattcgg aataccaagt acgagaagga 5580cggccagacg gtctacggga ccgacttcat tgccgataag gtggattatc tggacaccaa 5640ggcaccaggc gggtcaaatc aggaataagg gcacattgcc ccggcgtgag tcggggcaat 5700cccgcaagga gggtgaatga atcggacgtt tgaccggaag gcatacaggc aagaactgat 5760cgacgcgggg ttttccgccg aggatgccga aaccatcgca agccgcaccg tcatgcgtgc 5820gccccgcgaa accttccagt ccgtcggctc gatggtccag caagctacgg ccaagatcga 5880gcgcgacagc gtgcaactgg ctccccctgc cctgcccgcg ccatcggccg ccgtggagcg 5940ttcgcgtcgt ctcgaacagg aggcggcagg tttggcgaag tcgatgacca tcgacacgcg 6000aggaactatg acgaccaaga agcgaaaaac cgccggcgag gacctggcaa aacaggtcag 6060cgaggccaag caggccgcgt tgctgaaaca cacgaagcag cagatcaagg aaatgcagct 6120ttccttgttc gatattgcgc cgtggccgga cacgatgcga gcgatgccaa acgacacggc 6180ccgctctgcc ctgttcacca cgcgcaacaa gaaaatcccg cgcgaggcgc tgcaaaacaa 6240ggtcattttc cacgtcaaca aggacgtgaa gatcacctac accggcgtcg agctgcgggc 6300cgacgatgac gaactggtgt ggcagcaggt gttggagtac gcgaagcgca cccctatcgg 6360cgagccgatc accttcacgt tctacgagct ttgccaggac ctgggctggt cgatcaatgg 6420ccggtattac acgaaggccg aggaatgcct gtcgcgccta caggcgacgg cgatgggctt 6480cacgtccgac cgcgttgggc acctggaatc ggtgtcgctg ctgcaccgct tccgcgtcct 6540ggaccgtggc aagaaaacgt cccgttgcca ggtcctgatc gacgaggaaa tcgtcgtgct 6600gtttgctggc gaccactaca cgaaattcat atgggagaag taccgcaagc tgtcgccgac 6660ggcccgacgg atgttcgact atttcagctc gcaccgggag ccgtacccgc tcaagctgga 6720aaccttccgc ctcatgtgcg gatcggattc cacccgcgtg aagaagtggc gcgagcaggt 6780cggcgaagcc tgcgaagagt tgcgaggcag cggcctggtg gaacacgcct gggtcaatga 6840tgacctggtg cattgcaaac gctagggcct tgtggggtca gttccggctg ggggttcagc 6900agccagcgct ttactggcat ttcaggaaca agcgggcact gctcgacgca cttgcttcgc 6960tcagtatcgc tcgggacgca cggcgcgctc tacgaactgc cgataaacag aggattaaaa 7020ttgacaattg tgattaaggc tcagattcga cggcttggag cggccgacgt gcaggatttc 7080cgcgagatcc gattgtcggc cctgaagaaa gctccagaga tgttcgggtc cgtttacgag 7140cacgaggaga aaaagcccat ggaggcgttc gctgaacggt tgcgagatgc cgtggcattc 7200ggcgcctaca tcgacggcga gatcattggg ctgtcggtct tcaaacagga ggacggcccc 7260aaggacgctc acaaggcgca tctgtccggc gttttcgtgg agcccgaaca gcgaggccga 7320ggggtcgccg gtatgctgct gcgggcgttg ccggcgggtt tattgctcgt gatgatcgtc 7380cgacagattc caacgggaat ctggtggatg cgcatcttca tcctcggcgc acttaatatt 7440tcgctattct ggagcttgtt gtttatttcg gtctaccgcc tgccgggcgg ggtcgcggcg 7500acggtaggcg ctgtgcagcc gctgatggtc gtgttcatct ctgccgctct gctaggtagc 7560ccgatacgat tgatggcggt cctgggggct atttgcggaa ctgcgggcgt ggcgctgttg 7620gtgttgacac caaacgcagc gctagatcct gtcggcgtcg cagcgggcct ggcgggggcg 7680gtttccatgg cgttcggaac cgtgctgacc cgcaagtggc aacctcccgt gcctctgctc 7740acctttaccg cctggcaact ggcggccgga ggacttctgc tcgttccagt agctttagtg 7800tttgatccgc caatcccgat gcctacagga accaatgttc tcggcctggc gtggctcggc 7860ctgatcggag cgggtttaac ctacttcctt tggttccggg ggatctcgcg actcgaacct 7920acagttgttt ccttactggg ctttctcagc cccagatctg gggtcgatca gccggggatg 7980catcaggccg acagtcggaa cttcgggtcc ccgacctgta ccattcggtg agcaatggat 8040aggggagttg atatcgtcaa cgttcacttc taaagaaata gcgccactca gcttcctcag 8100cggctttatc cagcgatttc ctattatgtc ggcatagttc tcaagatcga cagcctgtca 8160cggttaagcg agaaatgaat aagaaggctg ataattcgga tctctgcgag ggagatgata 8220tttgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga 8280tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat 8340gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct 8400gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg 8460tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg 8520gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc aacagctgat 8580tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca 8640gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt ataaatcaaa 8700agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 8760gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 8820tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 8880ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 8940ggaagggaag aaagcgaaag gagcgggcgc cattcaggct gcgcaactgt tgggaagggc 9000gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 9060gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 9120aattaattcc catcttgaaa gaaatatagt ttaaatattt attgataaaa taacaagtca 9180ggtattatag tccaagcaaa aacataaatt tattgatgca agtttaaatt cagaaatatt 9240tcaataactg attatatcag ctggtacatt gccgtagatg aaagactgag tgcgatatta 9300tgtgtaatac ataaattgat gatatagcta gcttagctca tcgggggatc cgtcgaagct 9360agcttgggtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 9420tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 9480tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg 9540ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 9600tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 9660agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 9720gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 9780gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 9840gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 9900tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 9960agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga caggtcggtc 10020ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 10080ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 10140cctgcgtgca atccatcttg ttcaatccaa gctcccatgg gccctcgact agagtcgaga 10200tctggattga gagtgaatat gagactctaa ttggataccg aggggaattt atggaacgtc 10260agtggagcat ttttgacaag aaatatttgc tagctgatag tgaccttagg cgacttttga 10320acgcgcaata atggtttctg acgtatgtgc ttagctcatt aaactccaga aacccgcggc 10380tgagtggctc cttcaacgtt gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc 10440gtcatcggcg ggggtcataa cgtgactccc ttaattctcc gctcatgatc ttgatcccct 10500gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac 10560cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca 10620gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt 10680ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg 10740actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt gcttgcggca 10800gcgtgaagct tgcatgcctg caggtcgacg gcgcgccgag ctcctcgagc aaatttacac 10860attgccacta aacgtctaaa cccttgtaat ttgtttttgt tttactatgt gtgttatgta 10920tttgatttgc gataaatttt tatatttggt actaaattta taacaccttt tatgctaacg 10980tttgccaaca cttagcaatt tgcaagttga ttaattgatt ctaaattatt tttgtcttct 11040aaatacatat actaatcaac tggaaatgta aatatttgct aatatttcta ctataggaga 11100attaaagtga gtgaatatgg taccacaagg tttggagatt taattgttgc aatgctgcat 11160ggatggcata tacaccaaac attcaataat tcttgaggat aataatggta ccacacaaga 11220tttgaggtgc atgaacgtca cgtggacaaa aggtttagta atttttcaag acaacaatgt 11280taccacacac aagttttgag gtgcatgcat ggatgccctg tggaaagttt aaaaatattt 11340tggaaatgat ttgcatggaa gccatgtgta aaaccatgac atccacttgg aggatgcaat 11400aatgaagaaa actacaaatt tacatgcaac tagttatgca tgtagtctat ataatgagga 11460ttttgcaata ctttcattca tacacactca ctaagtttta cacgattata atttcttcat 11520agccagccca ccgcggtggg cggccgcctg cagtctagaa ggcctcctgc tttaatgaga 11580tatgcgagac gcctatgatc gcatgatatt tgctttcaat tctgttgtgc acgttgtaaa

11640aaacctgagc atgtgtagct cagatcctta ccgccggttt cggttcattc taatgaatat 11700atcacccgtt actatcgtat ttttatgaat aatattctcc gttcaattta ctgattgtcc 11760gtcgagcaaa tttacacatt gccactaaac gtctaaaccc ttgtaatttg tttttgtttt 11820actatgtgtg ttatgtattt gatttgcgat aaatttttat atttggtact aaatttataa 11880caccttttat gctaacgttt gccaacactt agcaatttgc aagttgatta attgattcta 11940aattattttt gtcttctaaa tacatatact aatcaactgg aaatgtaaat atttgctaat 12000atttctacta taggagaatt aaagtgagtg aatatggtac cacaaggttt ggagatttaa 12060ttgttgcaat gctgcatgga tggcatatac accaaacatt caataattct tgaggataat 12120aatggtacca cacaagattt gaggtgcatg aacgtcacgt ggacaaaagg tttagtaatt 12180tttcaagaca acaatgttac cacacacaag ttttgaggtg catgcatgga tgccctgtgg 12240aaagtttaaa aatattttgg aaatgatttg catggaagcc atgtgtaaaa ccatgacatc 12300cacttggagg atgcaataat gaagaaaact acaaatttac atgcaactag ttatgcatgt 12360agtctatata atgaggattt tgcaatactt tcattcatac acactcacta agttttacac 12420gattataatt tcttcatagc cagcggatcc gatatcgggc ccgctagcgt taaccctgct 12480ttaatgagat atgcgagacg cctatgatcg catgatattt gctttcaatt ctgttgtgca 12540cgttgtaaaa aacctgagca tgtgtagctc agatccttac cgccggtttc ggttcattct 12600aatgaatata tcacccgtta ctatcgtatt tttatgaata atattctccg ttcaatttac 12660tgattgtccg tcgagcaaat ttacacattg ccactaaacg tctaaaccct tgtaatttgt 12720ttttgtttta ctatgtgtgt tatgtatttg atttgcgata aatttttata tttggtacta 12780aatttataac accttttatg ctaacgtttg ccaacactta gcaatttgca agttgattaa 12840ttgattctaa attatttttg tcttctaaat acatatacta atcaactgga aatgtaaata 12900tttgctaata tttctactat aggagaatta aagtgagtga atatggtacc acaaggtttg 12960gagatttaat tgttgcaatg ctgcatggat ggcatataca ccaaacattc aataattctt 13020gaggataata atggtaccac acaagatttg aggtgcatga acgtcacgtg gacaaaaggt 13080ttagtaattt ttcaagacaa caatgttacc acacacaagt tttgaggtgc atgcatggat 13140gccctgtgga aagtttaaaa atattttgga aatgatttgc atggaagcca tgtgtaaaac 13200catgacatcc acttggagga tgcaataatg aagaaaacta caaatttaca tgcaactagt 13260tatgcatgta gtctatataa tgaggatttt gcaatacttt cattcataca cactcactaa 13320gttttacacg attataattt cttcatagcc agcagatctg ccggcatcga tcccgggcca 13380tggcctgctt taatgagata tgcgagacgc ctatgatcgc atgatatttg ctttcaattc 13440tgttgtgcac gttgtaaaaa acctgagcat gtgtagctca gatccttacc gccggtttcg 13500gttcattcta atgaatatat cacccgttac tatcgtattt ttatgaataa tattctccgt 13560tcaatttact gattgtccgt cgacgagctc ggcgcgcctc tagaggatcg atgaattcag 13620atcggctgag tggctccttc aacgttgcgg ttctgtcagt tccaaacgta aaacggcttg 13680tcccgcgtca tcggcggggg tcataacgtg actcccttaa ttctccgctc atgatcagat 13740tgtcgtttcc cgccttcagt ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 13800cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt gaaaaggttt 13860atccttcgtc catttgtatg tgcatgccaa ccacagggtt cccca 13905321443DNAPhaeodactylum tricornutumCDS(9)..(1442)delta-6-desaturase 32gatctaaa atg ggc aaa gga ggg gac gct cgg gcc tcg aag ggc tca acg 50 Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr 1 5 10 gcg gct cgc aag atc agt tgg cag gaa gtc aag acc cac gcg tct ccg 98Ala Ala Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro 15 20 25 30 gag gac gcc tgg atc att cac tcc aat aag gtc tac gac gtg tcc aac 146Glu Asp Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser Asn 35 40 45 tgg cac gaa cat ccc gga ggc gcc gtc att ttc acg cac gcc ggt gac 194Trp His Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly Asp 50 55 60 gac atg acg gac att ttc gct gcc ttt cac gca ccc gga tcg cag tcg 242Asp Met Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln Ser 65 70 75 ctc atg aag aag ttc tac att ggc gaa ttg ctc ccg gaa acc acc ggc 290Leu Met Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr Gly 80 85 90 aag gag ccg cag caa atc gcc ttt gaa aag ggc tac cgc gat ctg cgc 338Lys Glu Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu Arg 95 100 105 110 tcc aaa ctc atc atg atg ggc atg ttc aag tcc aac aag tgg ttc tac 386Ser Lys Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe Tyr 115 120 125 gtc tac aag tgc ctc agc aac atg gcc att tgg gcc gcc gcc tgt gct 434Val Tyr Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys Ala 130 135 140 ctc gtc ttt tac tcg gac cgc ttc tgg gta cac ctg gcc agc gcc gtc 482Leu Val Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala Val 145 150 155 atg ctg gga aca ttc ttt cag cag tcg gga tgg ttg gca cac gac ttt 530Met Leu Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp Phe 160 165 170 ctg cac cac cag gtc ttc acc aag cgc aag cac ggg gat ctc gga gga 578Leu His His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly Gly 175 180 185 190 ctc ttt tgg ggg aac ctc atg cag ggt tac tcc gta cag tgg tgg aaa 626Leu Phe Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp Lys 195 200 205 aac aag cac aac gga cac cac gcc gtc ccc aac ctc cac tgc tcc tcc 674Asn Lys His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser Ser 210 215 220 gca gtc gcg caa gat ggg gac ccg gac atc gat acc atg ccc ctt ctc 722Ala Val Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu 225 230 235 gcc tgg tcc gtc cag caa gcc cag tct tac cgg gaa ctc caa gcc gac 770Ala Trp Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp 240 245 250 gga aag gat tcg ggt ttg gtc aag ttc atg atc cgt aac caa tcc tac 818Gly Lys Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr 255 260 265 270 ttt tac ttt ccc atc ttg ttg ctc gcc cgc ctg tcg tgg ttg aac gag 866Phe Tyr Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu 275 280 285 tcc ttc aag tgc gcc ttt ggg ctt gga gct gcg tcg gag aac gct gct 914Ser Phe Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala 290 295 300 ctc gaa ctc aag gcc aag ggt ctt cag tac ccc ctt ttg gaa aag gct 962Leu Glu Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala 305 310 315 ggc atc ctg ctg cac tac gct tgg atg ctt aca gtt tcg tcc ggc ttt 1010Gly Ile Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe 320 325 330 gga cgc ttc tcg ttc gcg tac acc gca ttt tac ttt cta acc gcg acc 1058Gly Arg Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr 335 340 345 350 gcg tcc tgt gga ttc ttg ctc gcc att gtc ttt ggc ctc ggc cac aac 1106Ala Ser Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His Asn 355 360 365 ggc atg gcc acc tac aat gcc gac gcc cgt ccg gac ttc tgg aag ctc 1154Gly Met Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu 370 375 380 caa gtc acc acg act cgc aac gtc acg ggc gga cac ggt ttc ccc caa 1202Gln Val Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro Gln 385 390 395 gcc ttt gtc gac tgg ttc tgt ggt ggc ctc cag tac caa gtc gac cac 1250Ala Phe Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His 400 405 410 cac tta ttc ccc agc ctg ccc cga cac aat ctg gcc aag aca cac gca 1298His Leu Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His Ala 415 420 425 430 ctg gtc gaa tcg ttc tgc aag gag tgg ggt gtc cag tac cac gaa gcc 1346Leu Val Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala 435 440 445 gac ctt gtg gac ggg acc atg gaa gtc ttg cac cat ttg ggc agc gtg 1394Asp Leu Val Asp Gly Thr Met Glu Val Leu His His Leu Gly Ser Val 450 455 460 gcc ggc gaa ttc gtc gtg gat ttt gta cgc gat gga ccc gcc atg taa a 1443Ala Gly Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met 465 470 475 33477PRTPhaeodactylum tricornutum 33Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr Ala Ala 1 5 10 15 Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro Glu Asp 20 25 30 Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser Asn Trp His 35 40 45 Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly Asp Asp Met 50 55 60 Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln Ser Leu Met 65 70 75 80 Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr Gly Lys Glu 85 90 95 Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu Arg Ser Lys 100 105 110 Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe Tyr Val Tyr 115 120 125 Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys Ala Leu Val 130 135 140 Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala Val Met Leu 145 150 155 160 Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp Phe Leu His 165 170 175 His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly Gly Leu Phe 180 185 190 Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys 195 200 205 His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser Ser Ala Val 210 215 220 Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp 225 230 235 240 Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys 245 250 255 Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr 260 265 270 Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe 275 280 285 Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu 290 295 300 Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala Gly Ile 305 310 315 320 Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe Gly Arg 325 330 335 Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr Ala Ser 340 345 350 Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His Asn Gly Met 355 360 365 Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu Gln Val 370 375 380 Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro Gln Ala Phe 385 390 395 400 Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His His Leu 405 410 415 Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His Ala Leu Val 420 425 430 Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala Asp Leu 435 440 445 Val Asp Gly Thr Met Glu Val Leu His His Leu Gly Ser Val Ala Gly 450 455 460 Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met 465 470 475 3417061DNAArtificial sequenceConstruct comprising sequences encoding Physcomitrella patens delta-6-elongase, Caenorhabditis elegans LPLAT, and Phaeodactylum tricornutum delta-6-desaturase 34tggggaaccc tgtggttggc atgcacatac aaatggacga aggataaacc ttttcacgcc 60cttttaaata tccgattatt ctaataaacg ctcttttctc ttaggtttac ccgccaatat 120atcctgtcaa acactgatag tttaaactga aggcgggaaa cgacaatctg atcatgagcg 180gagaattaag ggagtcacgt tatgaccccc gccgatgacg cgggacaagc cgttttacgt 240ttggaactga cagaaccgca acgttgaagg agccactcag ccgatctgaa ttcatcgatc 300ctctagaggc gcgccgagct cctcgagcaa atttacacat tgccactaaa cgtctaaacc 360cttgtaattt gtttttgttt tactatgtgt gttatgtatt tgatttgcga taaattttta 420tatttggtac taaatttata acacctttta tgctaacgtt tgccaacact tagcaatttg 480caagttgatt aattgattct aaattatttt tgtcttctaa atacatatac taatcaactg 540gaaatgtaaa tatttgctaa tatttctact ataggagaat taaagtgagt gaatatggta 600ccacaaggtt tggagattta attgttgcaa tgctgcatgg atggcatata caccaaacat 660tcaataattc ttgaggataa taatggtacc acacaagatt tgaggtgcat gaacgtcacg 720tggacaaaag gtttagtaat ttttcaagac aacaatgtta ccacacacaa gttttgaggt 780gcatgcatgg atgccctgtg gaaagtttaa aaatattttg gaaatgattt gcatggaagc 840catgtgtaaa accatgacat ccacttggag gatgcaataa tgaagaaaac tacaaattta 900catgcaacta gttatgcatg tagtctatat aatgaggatt ttgcaatact ttcattcata 960cacactcact aagttttaca cgattataat ttcttcatag ccagcccacc gcggtgggcg 1020gccgc atg gag gtc gtg gag aga ttc tac ggt gag ttg gat ggg aag gtc 1070 Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val 1 5 10 15 tcg cag ggc gtg aat gca ttg ctg ggt agt ttt ggg gtg gag ttg acg 1118Ser Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr 20 25 30 gat acg ccc act acc aaa ggc ttg ccc ctc gtt gac agt ccc aca ccc 1166Asp Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro 35 40 45 atc gtc ctc ggt gtt tct gta tac ttg act att gtc att gga ggg ctt 1214Ile Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu 50 55 60 ttg tgg ata aag gcc agg gat ctg aaa ccg cgc gcc tcg gag cca ttt 1262Leu Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe 65 70 75 ttg ctc caa gct ttg gtg ctt gtg cac aac ctg ttc tgt ttt gcg ctc 1310Leu Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu 80 85 90 95 agt ctg tat atg tgc gtg ggc atc gct tat cag gct att acc tgg cgg 1358Ser Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg 100 105 110 tac tct ctc tgg ggc aat gca tac aat cct aaa cat aaa gag atg gcg 1406Tyr Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala 115 120 125 att ctg gta tac ttg ttc tac atg tct aag tac gtg gaa ttc atg gat 1454Ile Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp 130 135 140 acc gtt atc atg ata ctg aag cgc agc acc agg caa ata agc ttc ctc 1502Thr Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu 145 150 155 cac gtt tat cat cat tct tca att tcc ctc att tgg tgg gct att gct 1550His Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala 160 165 170 175 cat cac gct cct ggc ggt gaa gca tat tgg tct gcg gct ctg aac tca 1598His His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser 180 185 190 gga gtg cat gtt ctc atg tat gcg tat tac ttc ttg gct gcc tgc ctt 1646Gly Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu 195 200 205 cga agt agc cca aag tta aaa aat aag tac ctt ttt tgg ggc agg tac 1694Arg Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr 210 215 220 ttg aca caa ttc caa atg ttc cag ttt atg ctg aac tta gtg cag gct 1742Leu Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala 225 230 235 tac tac gac atg aaa acg aat gcg cca tat cca caa tgg ctg atc aag 1790Tyr Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys 240 245 250 255 att ttg ttc tac tac atg atc tcg ttg ctg ttt ctt ttc ggc aat ttt 1838Ile Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe

260 265 270 tac gta caa aaa tac atc aaa ccc tct gac gga aag caa aag gga gct 1886Tyr Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala 275 280 285 aaa act gag tga tctagaaggc ctcctgcttt aatgagatat gcgagacgcc 1938Lys Thr Glu 290 tatgatcgca tgatatttgc tttcaattct gttgtgcacg ttgtaaaaaa cctgagcatg 1998tgtagctcag atccttaccg ccggtttcgg ttcattctaa tgaatatatc acccgttact 2058atcgtatttt tatgaataat attctccgtt caatttactg attgtccgtc gagcaaattt 2118acacattgcc actaaacgtc taaacccttg taatttgttt ttgttttact atgtgtgtta 2178tgtatttgat ttgcgataaa tttttatatt tggtactaaa tttataacac cttttatgct 2238aacgtttgcc aacacttagc aatttgcaag ttgattaatt gattctaaat tatttttgtc 2298ttctaaatac atatactaat caactggaaa tgtaaatatt tgctaatatt tctactatag 2358gagaattaaa gtgagtgaat atggtaccac aaggtttgga gatttaattg ttgcaatgct 2418gcatggatgg catatacacc aaacattcaa taattcttga ggataataat ggtaccacac 2478aagatttgag gtgcatgaac gtcacgtgga caaaaggttt agtaattttt caagacaaca 2538atgttaccac acacaagttt tgaggtgcat gcatggatgc cctgtggaaa gtttaaaaat 2598attttggaaa tgatttgcat ggaagccatg tgtaaaacca tgacatccac ttggaggatg 2658caataatgaa gaaaactaca aatttacatg caactagtta tgcatgtagt ctatataatg 2718aggattttgc aatactttca ttcatacaca ctcactaagt tttacacgat tataatttct 2778tcatagccag cggatccgcc cacata atg gag aac ttc tgg tct att gtt gtg 2831 Met Glu Asn Phe Trp Ser Ile Val Val 295 ttt ttt cta ctc tca att ctc ttc att tta tat aac ata tcg aca gta 2879Phe Phe Leu Leu Ser Ile Leu Phe Ile Leu Tyr Asn Ile Ser Thr Val 300 305 310 315 tgc cac tac tat atg cgg att tcg ttt tat tac ttc aca att tta ttg 2927Cys His Tyr Tyr Met Arg Ile Ser Phe Tyr Tyr Phe Thr Ile Leu Leu 320 325 330 cat gga atg gaa gtt tgt gtt aca atg atc cct tct tgg cta aat ggg 2975His Gly Met Glu Val Cys Val Thr Met Ile Pro Ser Trp Leu Asn Gly 335 340 345 aag ggt gct gat tac gtg ttt cac tcg ttt ttc tat tgg tgt aaa tgg 3023Lys Gly Ala Asp Tyr Val Phe His Ser Phe Phe Tyr Trp Cys Lys Trp 350 355 360 act ggt gtt cat aca aca gtc tat gga tat gaa aaa aca caa gtt gaa 3071Thr Gly Val His Thr Thr Val Tyr Gly Tyr Glu Lys Thr Gln Val Glu 365 370 375 ggt ccg gct gta gtt att tgt aat cat cag agt tct ctc gac att cta 3119Gly Pro Ala Val Val Ile Cys Asn His Gln Ser Ser Leu Asp Ile Leu 380 385 390 395 tcg atg gca tca atc tgg ccg aag aat tgt gtt gta atg atg aaa cga 3167Ser Met Ala Ser Ile Trp Pro Lys Asn Cys Val Val Met Met Lys Arg 400 405 410 att ctt gcc tat gtt cca ttc ttc aat ctc gga gcc tac ttt tcc aac 3215Ile Leu Ala Tyr Val Pro Phe Phe Asn Leu Gly Ala Tyr Phe Ser Asn 415 420 425 aca atc ttc atc gat cga tat aac cgt gaa cgt gcg atg gct tca gtt 3263Thr Ile Phe Ile Asp Arg Tyr Asn Arg Glu Arg Ala Met Ala Ser Val 430 435 440 gat tat tgt gca tct gaa atg aag aac aga aat ctt aaa ctt tgg gta 3311Asp Tyr Cys Ala Ser Glu Met Lys Asn Arg Asn Leu Lys Leu Trp Val 445 450 455 ttt ccg gaa gga aca aga aat cgt gaa gga ggg ttc att cca ttc aag 3359Phe Pro Glu Gly Thr Arg Asn Arg Glu Gly Gly Phe Ile Pro Phe Lys 460 465 470 475 aaa gga gca ttc aat att gca gtt cgt gcg cag att ccc att att cca 3407Lys Gly Ala Phe Asn Ile Ala Val Arg Ala Gln Ile Pro Ile Ile Pro 480 485 490 gtt gta ttc tca gac tat cgg gat ttc tac tca aag cca ggc cga tat 3455Val Val Phe Ser Asp Tyr Arg Asp Phe Tyr Ser Lys Pro Gly Arg Tyr 495 500 505 ttc aag aat gat gga gaa gtt gtt att cga gtt ctg gat gcg att cca 3503Phe Lys Asn Asp Gly Glu Val Val Ile Arg Val Leu Asp Ala Ile Pro 510 515 520 aca aaa ggg ctc act ctt gat gac gtc agc gag ttg tct gat atg tgt 3551Thr Lys Gly Leu Thr Leu Asp Asp Val Ser Glu Leu Ser Asp Met Cys 525 530 535 cgg gac gtt atg ttg gca gcc tat aag gaa gtt act cta gaa gct cag 3599Arg Asp Val Met Leu Ala Ala Tyr Lys Glu Val Thr Leu Glu Ala Gln 540 545 550 555 caa cga aat gcg aca cgg cgt gga gaa aca aaa gac ggg aag aaa tct 3647Gln Arg Asn Ala Thr Arg Arg Gly Glu Thr Lys Asp Gly Lys Lys Ser 560 565 570 gag taa gctagcgtta accctgcttt aatgagatat gcgagacgcc tatgatcgca 3703Glu tgatatttgc tttcaattct gttgtgcacg ttgtaaaaaa cctgagcatg tgtagctcag 3763atccttaccg ccggtttcgg ttcattctaa tgaatatatc acccgttact atcgtatttt 3823tatgaataat attctccgtt caatttactg attgtccgtc gagcaaattt acacattgcc 3883actaaacgtc taaacccttg taatttgttt ttgttttact atgtgtgtta tgtatttgat 3943ttgcgataaa tttttatatt tggtactaaa tttataacac cttttatgct aacgtttgcc 4003aacacttagc aatttgcaag ttgattaatt gattctaaat tatttttgtc ttctaaatac 4063atatactaat caactggaaa tgtaaatatt tgctaatatt tctactatag gagaattaaa 4123gtgagtgaat atggtaccac aaggtttgga gatttaattg ttgcaatgct gcatggatgg 4183catatacacc aaacattcaa taattcttga ggataataat ggtaccacac aagatttgag 4243gtgcatgaac gtcacgtgga caaaaggttt agtaattttt caagacaaca atgttaccac 4303acacaagttt tgaggtgcat gcatggatgc cctgtggaaa gtttaaaaat attttggaaa 4363tgatttgcat ggaagccatg tgtaaaacca tgacatccac ttggaggatg caataatgaa 4423gaaaactaca aatttacatg caactagtta tgcatgtagt ctatataatg aggattttgc 4483aatactttca ttcatacaca ctcactaagt tttacacgat tataatttct tcatagccag 4543cagatctaaa atg ggc aaa gga ggg gac gct cgg gcc tcg aag ggc tca 4592 Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser 575 580 585 acg gcg gct cgc aag atc agt tgg cag gaa gtc aag acc cac gcg tct 4640Thr Ala Ala Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser 590 595 600 ccg gag gac gcc tgg atc att cac tcc aat aag gtc tac gac gtg tcc 4688Pro Glu Asp Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser 605 610 615 aac tgg cac gaa cat ccc gga ggc gcc gtc att ttc acg cac gcc ggt 4736Asn Trp His Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly 620 625 630 gac gac atg acg gac att ttc gct gcc ttt cac gca ccc gga tcg cag 4784Asp Asp Met Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln 635 640 645 tcg ctc atg aag aag ttc tac att ggc gaa ttg ctc ccg gaa acc acc 4832Ser Leu Met Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr 650 655 660 665 ggc aag gag ccg cag caa atc gcc ttt gaa aag ggc tac cgc gat ctg 4880Gly Lys Glu Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu 670 675 680 cgc tcc aaa ctc atc atg atg ggc atg ttc aag tcc aac aag tgg ttc 4928Arg Ser Lys Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe 685 690 695 tac gtc tac aag tgc ctc agc aac atg gcc att tgg gcc gcc gcc tgt 4976Tyr Val Tyr Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys 700 705 710 gct ctc gtc ttt tac tcg gac cgc ttc tgg gta cac ctg gcc agc gcc 5024Ala Leu Val Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala 715 720 725 gtc atg ctg gga aca ttc ttt cag cag tcg gga tgg ttg gca cac gac 5072Val Met Leu Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp 730 735 740 745 ttt ctg cac cac cag gtc ttc acc aag cgc aag cac ggg gat ctc gga 5120Phe Leu His His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly 750 755 760 gga ctc ttt tgg ggg aac ctc atg cag ggt tac tcc gta cag tgg tgg 5168Gly Leu Phe Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp 765 770 775 aaa aac aag cac aac gga cac cac gcc gtc ccc aac ctc cac tgc tcc 5216Lys Asn Lys His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser 780 785 790 tcc gca gtc gcg caa gat ggg gac ccg gac atc gat acc atg ccc ctt 5264Ser Ala Val Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu 795 800 805 ctc gcc tgg tcc gtc cag caa gcc cag tct tac cgg gaa ctc caa gcc 5312Leu Ala Trp Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala 810 815 820 825 gac gga aag gat tcg ggt ttg gtc aag ttc atg atc cgt aac caa tcc 5360Asp Gly Lys Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser 830 835 840 tac ttt tac ttt ccc atc ttg ttg ctc gcc cgc ctg tcg tgg ttg aac 5408Tyr Phe Tyr Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn 845 850 855 gag tcc ttc aag tgc gcc ttt ggg ctt gga gct gcg tcg gag aac gct 5456Glu Ser Phe Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala 860 865 870 gct ctc gaa ctc aag gcc aag ggt ctt cag tac ccc ctt ttg gaa aag 5504Ala Leu Glu Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys 875 880 885 gct ggc atc ctg ctg cac tac gct tgg atg ctt aca gtt tcg tcc ggc 5552Ala Gly Ile Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly 890 895 900 905 ttt gga cgc ttc tcg ttc gcg tac acc gca ttt tac ttt cta acc gcg 5600Phe Gly Arg Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala 910 915 920 acc gcg tcc tgt gga ttc ttg ctc gcc att gtc ttt ggc ctc ggc cac 5648Thr Ala Ser Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His 925 930 935 aac ggc atg gcc acc tac aat gcc gac gcc cgt ccg gac ttc tgg aag 5696Asn Gly Met Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys 940 945 950 ctc caa gtc acc acg act cgc aac gtc acg ggc gga cac ggt ttc ccc 5744Leu Gln Val Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro 955 960 965 caa gcc ttt gtc gac tgg ttc tgt ggt ggc ctc cag tac caa gtc gac 5792Gln Ala Phe Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp 970 975 980 985 cac cac tta ttc ccc agc ctg ccc cga cac aat ctg gcc aag aca cac 5840His His Leu Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His 990 995 1000 gca ctg gtc gaa tcg ttc tgc aag gag tgg ggt gtc cag tac cac 5885Ala Leu Val Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His 1005 1010 1015 gaa gcc gac ctt gtg gac ggg acc atg gaa gtc ttg cac cat ttg 5930Glu Ala Asp Leu Val Asp Gly Thr Met Glu Val Leu His His Leu 1020 1025 1030 ggc agc gtg gcc ggc gaa ttc gtc gtg gat ttt gta cgc gat gga 5975Gly Ser Val Ala Gly Glu Phe Val Val Asp Phe Val Arg Asp Gly 1035 1040 1045 ccc gcc atg taa agatctgccg gcatcgatcc cgggccatgg cctgctttaa 6027Pro Ala Met tgagatatgc gagacgccta tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt 6087gtaaaaaacc tgagcatgtg tagctcagat ccttaccgcc ggtttcggtt cattctaatg 6147aatatatcac ccgttactat cgtattttta tgaataatat tctccgttca atttactgat 6207tgtccgtcga cgagctcggc gcgccgtcga cctgcaggca tgcaagcttc acgctgccgc 6267aagcactcag ggcgcaaggg ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag 6327aaacggtgct gaccccggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 6387agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 6447gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 6507aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 6567tcaagatcat gagcggagaa ttaagggagt cacgttatga cccccgccga tgacgcggga 6627caagccgttt tacgtttgga actgacagaa ccgcaacgtt gaaggagcca ctcagccgcg 6687ggtttctgga gtttaatgag ctaagcacat acgtcagaaa ccattattgc gcgttcaaaa 6747gtcgcctaag gtcactatca gctagcaaat atttcttgtc aaaaatgctc cactgacgtt 6807ccataaattc ccctcggtat ccaattagag tctcatattc actctcaatc cagatctcga 6867ctctagtcga gggcccatgg gagcttggat tgaacaagat ggattgcacg caggttctcc 6927ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 6987tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 7047cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac 7107gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 7167gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 7227agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 7287attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 7347tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 7407caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 7467cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 7527gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 7587tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 7647gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggaccca agctagcttc 7707gacggatccc ccgatgagct aagctagcta tatcatcaat ttatgtatta cacataatat 7767cgcactcagt ctttcatcta cggcaatgta ccagctgata taatcagtta ttgaaatatt 7827tctgaattta aacttgcatc aataaattta tgtttttgct tggactataa tacctgactt 7887gttattttat caataaatat ttaaactata tttctttcaa gatgggaatt aattcactgg 7947ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 8007cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 8067cccaacagtt gcgcagcctg aatggcgccc gctcctttcg ctttcttccc ttcctttctc 8127gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 8187tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt 8247gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 8307agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat 8367ttataaggga ttttgccgat ttcggaacca ccatcaaaca ggattttcgc ctgctggggc 8427aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc 8487tgttgcccgt ctcactggtg aaaagaaaaa ccaccccagt acattaaaaa cgtccgcaat 8547gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatcct gccaccagcc 8607agccaacagc tccccgaccg gcagctcggc acaaaatcac cactcgatac aggcagccca 8667tcagtccggg acggcgtcag cgggagagcc gttgtaaggc ggcagacttt gctcatgtta 8727ccgatgctat tcggaagaac ggcaactaag ctgccgggtt tgaaacacgg atgatctcgc 8787ggagggtagc atgttgattg taacgatgac agagcgttgc tgcctgtgat caaatatcat 8847ctccctcgca gagatccgaa ttatcagcct tcttattcat ttctcgctta accgtgacag 8907gctgtcgatc ttgagaacta tgccgacata ataggaaatc gctggataaa gccgctgagg 8967aagctgagtg gcgctatttc tttagaagtg aacgttgacg atatcaactc ccctatccat 9027tgctcaccga atggtacagg tcggggaccc gaagttccga ctgtcggcct gatgcatccc 9087cggctgatcg accccagatc tggggctgag aaagcccagt aaggaaacaa ctgtaggttc 9147gagtcgcgag atcccccgga accaaaggaa gtaggttaaa cccgctccga tcaggccgag 9207ccacgccagg ccgagaacat tggttcctgt aggcatcggg attggcggat caaacactaa 9267agctactgga acgagcagaa gtcctccggc cgccagttgc caggcggtaa aggtgagcag 9327aggcacggga ggttgccact tgcgggtcag cacggttccg aacgccatgg aaaccgcccc 9387cgccaggccc gctgcgacgc cgacaggatc tagcgctgcg tttggtgtca acaccaacag 9447cgccacgccc gcagttccgc aaatagcccc caggaccgcc atcaatcgta tcgggctacc 9507tagcagagcg gcagagatga acacgaccat cagcggctgc acagcgccta ccgtcgccgc 9567gaccccgccc ggcaggcggt agaccgaaat aaacaacaag ctccagaata gcgaaatatt 9627aagtgcgccg aggatgaaga tgcgcatcca ccagattccc gttggaatct gtcggacgat 9687catcacgagc aataaacccg ccggcaacgc ccgcagcagc ataccggcga cccctcggcc 9747tcgctgttcg ggctccacga aaacgccgga cagatgcgcc ttgtgagcgt ccttggggcc 9807gtcctcctgt ttgaagaccg acagcccaat gatctcgccg tcgatgtagg cgccgaatgc 9867cacggcatct cgcaaccgtt cagcgaacgc ctccatgggc tttttctcct cgtgctcgta 9927aacggacccg aacatctctg gagctttctt cagggccgac aatcggatct cgcggaaatc 9987ctgcacgtcg gccgctccaa gccgtcgaat ctgagcctta atcacaattg tcaattttaa 10047tcctctgttt atcggcagtt cgtagagcgc gccgtgcgtc ccgagcgata ctgagcgaag 10107caagtgcgtc gagcagtgcc cgcttgttcc tgaaatgcca gtaaagcgct ggctgctgaa 10167cccccagccg gaactgaccc cacaaggccc tagcgtttgc aatgcaccag

gtcatcattg 10227acccaggcgt gttccaccag gccgctgcct cgcaactctt cgcaggcttc gccgacctgc 10287tcgcgccact tcttcacgcg ggtggaatcc gatccgcaca tgaggcggaa ggtttccagc 10347ttgagcgggt acggctcccg gtgcgagctg aaatagtcga acatccgtcg ggccgtcggc 10407gacagcttgc ggtacttctc ccatatgaat ttcgtgtagt ggtcgccagc aaacagcacg 10467acgatttcct cgtcgatcag gacctggcaa cgggacgttt tcttgccacg gtccaggacg 10527cggaagcggt gcagcagcga caccgattcc aggtgcccaa cgcggtcgga cgtgaagccc 10587atcgccgtcg cctgtaggcg cgacaggcat tcctcggcct tcgtgtaata ccggccattg 10647atcgaccagc ccaggtcctg gcaaagctcg tagaacgtga aggtgatcgg ctcgccgata 10707ggggtgcgct tcgcgtactc caacacctgc tgccacacca gttcgtcatc gtcggcccgc 10767agctcgacgc cggtgtaggt gatcttcacg tccttgttga cgtggaaaat gaccttgttt 10827tgcagcgcct cgcgcgggat tttcttgttg cgcgtggtga acagggcaga gcgggccgtg 10887tcgtttggca tcgctcgcat cgtgtccggc cacggcgcaa tatcgaacaa ggaaagctgc 10947atttccttga tctgctgctt cgtgtgtttc agcaacgcgg cctgcttggc ctcgctgacc 11007tgttttgcca ggtcctcgcc ggcggttttt cgcttcttgg tcgtcatagt tcctcgcgtg 11067tcgatggtca tcgacttcgc caaacctgcc gcctcctgtt cgagacgacg cgaacgctcc 11127acggcggccg atggcgcggg cagggcaggg ggagccagtt gcacgctgtc gcgctcgatc 11187ttggccgtag cttgctggac catcgagccg acggactgga aggtttcgcg gggcgcacgc 11247atgacggtgc ggcttgcgat ggtttcggca tcctcggcgg aaaaccccgc gtcgatcagt 11307tcttgcctgt atgccttccg gtcaaacgtc cgattcattc accctccttg cgggattgcc 11367ccgactcacg ccggggcaat gtgcccttat tcctgatttg acccgcctgg tgccttggtg 11427tccagataat ccaccttatc ggcaatgaag tcggtcccgt agaccgtctg gccgtccttc 11487tcgtacttgg tattccgaat cttgccctgc acgaatacca gcgacccctt gcccaaatac 11547ttgccgtggg cctcggcctg agagccaaaa cacttgatgc ggaagaagtc ggtgcgctcc 11607tgcttgtcgc cggcatcgtt gcgccacatc taggtactaa aacaattcat ccagtaaaat 11667ataatatttt attttctccc aatcaggctt gatccccagt aagtcaaaaa atagctcgac 11727atactgttct tccccgatat cctccctgat cgaccggacg cagaaggcaa tgtcatacca 11787cttgtccgcc ctgccgcttc tcccaagatc aataaagcca cttactttgc catctttcac 11847aaagatgttg ctgtctccca ggtcgccgtg ggaaaagaca agttcctctt cgggcttttc 11907cgtctttaaa aaatcataca gctcgcgcgg atctttaaat ggagtgtctt cttcccagtt 11967ttcgcaatcc acatcggcca gatcgttatt cagtaagtaa tccaattcgg ctaagcggct 12027gtctaagcta ttcgtatagg gacaatccga tatgtcgatg gagtgaaaga gcctgatgca 12087ctccgcatac agctcgataa tcttttcagg gctttgttca tcttcatact cttccgagca 12147aaggacgcca tcggcctcac tcatgagcag attgctccag ccatcatgcc gttcaaagtg 12207caggaccttt ggaacaggca gctttccttc cagccatagc atcatgtcct tttcccgttc 12267cacatcatag gtggtccctt tataccggct gtccgtcatt tttaaatata ggttttcatt 12327ttctcccacc agcttatata ccttagcagg agacattcct tccgtatctt ttacgcagcg 12387gtatttttcg atcagttttt tcaattccgg tgatattctc attttagcca tttattattt 12447ccttcctctt ttctacagta tttaaagata ccccaagaag ctaattataa caagacgaac 12507tccaattcac tgttccttgc attctaaaac cttaaatacc agaaaacagc tttttcaaag 12567ttgttttcaa agttggcgta taacatagta tcgacggagc cgattttgaa accacaatta 12627tgggtgatgc tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg 12687gcttctgtgt ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc 12747aaaagcaccg ccggacatca gcgctatctc tgctctcact gccgtaaaac atggcaactg 12807cagttcactt acaccgcttc tcaacccggt acgcaccaga aaatcattga tatggccatg 12867aatggcgttg gatgccgggc aacagcccgc attatgggcg ttggcctcaa cacgatttta 12927cgtcacttaa aaaactcagg ccgcagtcgg taacctcgcg catacagccg ggcagtgacg 12987tcatcgtctg cgcggaaatg gacgaacagt ggggctatgt cggggctaaa tcgcgccagc 13047gctggctgtt ttacgcgtat gacagtctcc ggaagacggt tgttgcgcac gtattcggtg 13107aacgcactat ggcgacgctg gggcgtctta tgagcctgct gtcacccttt gacgtggtga 13167tatggatgac ggatggctgg ccgctgtatg aatcccgcct gaagggaaag ctgcacgtaa 13227tcagcaagcg atatacgcag cgaattgagc ggcataacct gaatctgagg cagcacctgg 13287cacggctggg acggaagtcg ctgtcgttct caaaatcggt ggagctgcat gacaaagtca 13347tcgggcatta tctgaacata aaacactatc aataagttgg agtcattacc caattatgat 13407agaatttaca agctataagg ttattgtcct gggtttcaag cattagtcca tgcaagtttt 13467tatgctttgc ccattctata gatatattga taagcgcgct gcctatgcct tgccccctga 13527aatccttaca tacggcgata tcttctatat aaaagatata ttatcttatc agtattgtca 13587atatattcaa ggcaatctgc ctcctcatcc tcttcatcct cttcgtcttg gtagcttttt 13647aaatatggcg cttcatagag taattctgta aaggtccaat tctcgttttc atacctcggt 13707ataatcttac ctatcacctc aaatggttcg ctgggtttat cgcacccccg aacacgagca 13767cggcacccgc gaccactatg ccaagaatgc ccaaggtaaa aattgccggc cccgccatga 13827agtccgtgaa tgccccgacg gccgaagtga agggcaggcc gccacccagg ccgccgccct 13887cactgcccgg cacctggtcg ctgaatgtcg atgccagcac ctgcggcacg tcaatgcttc 13947cgggcgtcgc gctcgggctg atcgcccatc ccgttactgc cccgatcccg gcaatggcaa 14007ggactgccag cgctgccatt tttggggtga ggccgttcgc ggccgagggg cgcagcccct 14067ggggggatgg gaggcccgcg ttagcgggcc gggagggttc gagaaggggg ggcacccccc 14127ttcggcgtgc gcggtcacgc gcacagggcg cagccctggt taaaaacaag gtttataaat 14187attggtttaa aagcaggtta aaagacaggt tagcggtggc cgaaaaacgg gcggaaaccc 14247ttgcaaatgc tggattttct gcctgtggac agcccctcaa atgtcaatag gtgcgcccct 14307catctgtcag cactctgccc ctcaagtgtc aaggatcgcg cccctcatct gtcagtagtc 14367gcgcccctca agtgtcaata ccgcagggca cttatcccca ggcttgtcca catcatctgt 14427gggaaactcg cgtaaaatca ggcgttttcg ccgatttgcg aggctggcca gctccacgtc 14487gccggccgaa atcgagcctg cccctcatct gtcaacgccg cgccgggtga gtcggcccct 14547caagtgtcaa cgtccgcccc tcatctgtca gtgagggcca agttttccgc gaggtatcca 14607caacgccggc ggccgcggtg tctcgcacac ggcttcgacg gcgtttctgg cgcgtttgca 14667gggccataga cggccgccag cccagcggcg agggcaacca gcccggtgag cgtcgcaaag 14727gcgctcggtc ttgccttgct cgtcggtgat gtacttcacc agctccgcga agtcgctctt 14787cttgatggag cgcatgggga cgtgcttggc aatcacgcgc accccccggc cgttttagcg 14847gctaaaaaag tcatggctct gccctcgggc ggaccacgcc catcatgacc ttgccaagct 14907cgtcctgctt ctcttcgatc ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg 14967ccgtgcgcgg gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc 15027cattgatgcg ggccagctcg cggacgtgct catagtccac gacgcccgtg attttgtagc 15087cctggccgac ggccagcagg taggccgaca ggctcatgcc ggccgccgcc gccttttcct 15147caatcgctct tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg 15207ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc 15267agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga 15327aggaacaccc gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga 15387tacaccaagg aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg 15447atggatatac cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaagc 15507gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 15567ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 15627tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 15687tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 15747cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 15807tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 15867gcggaagagc gccagaaggc cgccagagag gccgagcgcg gccgtgaggc ttggacgcta 15927gggcagggca tgaaaaagcc cgtagcgggc tgctacgggc gtctgacgcg gtggaaaggg 15987ggaggggatg ttgtctacat ggctctgctg tagtgagtgg gttgcgctcc ggcagcggtc 16047ctgatcaatc gtcacccttt ctcggtcctt caacgttcct gacaacgagc ctccttttcg 16107ccaatccatc gacaatcacc gcgagtccct gctcgaacgc tgcgtccgga ccggcttcgt 16167cgaaggcgtc tatcgcggcc cgcaacagcg gcgagagcgg agcctgttca acggtgccgc 16227cgcgctcgcc ggcatcgctg tcgccggcct gctcctcaag cacggcccca acagtgaagt 16287agctgattgt catcagcgca ttgacggcgt ccccggccga aaaacccgcc tcgcagagga 16347agcgaagctg cgcgtcggcc gtttccatct gcggtgcgcc cggtcgcgtg ccggcatgga 16407tgcgcgcgcc atcgcggtag gcgagcagcg cctgcctgaa gctgcgggca ttcccgatca 16467gaaatgagcg ccagtcgtcg tcggctctcg gcaccgaatg cgtatgattc tccgccagca 16527tggcttcggc cagtgcgtcg agcagcgccc gcttgttcct gaagtgccag taaagcgccg 16587gctgctgaac ccccaaccgt tccgccagtt tgcgtgtcgt cagaccgtct acgccgacct 16647cgttcaacag gtccagggcg gcacggatca ctgtattcgg ctgcaacttt gtcatgcttg 16707acactttatc actgataaac ataatatgtc caccaactta tcagtgataa agaatccgcg 16767cgttcaatcg gaccagcgga ggctggtccg gaggccagac gtgaaaccca acatacccct 16827gatcgtaatt ctgagcactg tcgcgctcga cgctgtcggc atcggcctga ttatgccggt 16887gctgccgggc ctcctgcgcg atctggttca ctcgaacgac gtcaccgccc actatggcat 16947tctgctggcg ctgtatgcgt tggtgcaatt tgcctgcgca cctgtgctgg gcgcgctgtc 17007ggatcgtttc gggcggcggc caatcttgct cgtctcgctg gccggcgcca gatc 1706135290PRTArtificial sequencePhyscomitrella patens delta-6-elongase 35Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser 1 5 10 15 Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp 20 25 30 Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile 35 40 45 Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu 50 55 60 Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu 65 70 75 80 Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser 85 90 95 Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr 100 105 110 Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile 115 120 125 Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr 130 135 140 Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His 145 150 155 160 Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His 165 170 175 His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly 180 185 190 Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg 195 200 205 Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu 210 215 220 Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr 225 230 235 240 Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile 245 250 255 Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr 260 265 270 Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys 275 280 285 Thr Glu 290 36282PRTArtificial sequenceCaenorhabditis elegans LPLAT 36Met Glu Asn Phe Trp Ser Ile Val Val Phe Phe Leu Leu Ser Ile Leu 1 5 10 15 Phe Ile Leu Tyr Asn Ile Ser Thr Val Cys His Tyr Tyr Met Arg Ile 20 25 30 Ser Phe Tyr Tyr Phe Thr Ile Leu Leu His Gly Met Glu Val Cys Val 35 40 45 Thr Met Ile Pro Ser Trp Leu Asn Gly Lys Gly Ala Asp Tyr Val Phe 50 55 60 His Ser Phe Phe Tyr Trp Cys Lys Trp Thr Gly Val His Thr Thr Val 65 70 75 80 Tyr Gly Tyr Glu Lys Thr Gln Val Glu Gly Pro Ala Val Val Ile Cys 85 90 95 Asn His Gln Ser Ser Leu Asp Ile Leu Ser Met Ala Ser Ile Trp Pro 100 105 110 Lys Asn Cys Val Val Met Met Lys Arg Ile Leu Ala Tyr Val Pro Phe 115 120 125 Phe Asn Leu Gly Ala Tyr Phe Ser Asn Thr Ile Phe Ile Asp Arg Tyr 130 135 140 Asn Arg Glu Arg Ala Met Ala Ser Val Asp Tyr Cys Ala Ser Glu Met 145 150 155 160 Lys Asn Arg Asn Leu Lys Leu Trp Val Phe Pro Glu Gly Thr Arg Asn 165 170 175 Arg Glu Gly Gly Phe Ile Pro Phe Lys Lys Gly Ala Phe Asn Ile Ala 180 185 190 Val Arg Ala Gln Ile Pro Ile Ile Pro Val Val Phe Ser Asp Tyr Arg 195 200 205 Asp Phe Tyr Ser Lys Pro Gly Arg Tyr Phe Lys Asn Asp Gly Glu Val 210 215 220 Val Ile Arg Val Leu Asp Ala Ile Pro Thr Lys Gly Leu Thr Leu Asp 225 230 235 240 Asp Val Ser Glu Leu Ser Asp Met Cys Arg Asp Val Met Leu Ala Ala 245 250 255 Tyr Lys Glu Val Thr Leu Glu Ala Gln Gln Arg Asn Ala Thr Arg Arg 260 265 270 Gly Glu Thr Lys Asp Gly Lys Lys Ser Glu 275 280 37477PRTArtificial sequencePhaeodactylum tricornutum delta-6-desaturase 37Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr Ala Ala 1 5 10 15 Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro Glu Asp 20 25 30 Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser Asn Trp His 35 40 45 Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly Asp Asp Met 50 55 60 Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln Ser Leu Met 65 70 75 80 Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr Gly Lys Glu 85 90 95 Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu Arg Ser Lys 100 105 110 Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe Tyr Val Tyr 115 120 125 Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys Ala Leu Val 130 135 140 Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala Val Met Leu 145 150 155 160 Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp Phe Leu His 165 170 175 His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly Gly Leu Phe 180 185 190 Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys 195 200 205 His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser Ser Ala Val 210 215 220 Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp 225 230 235 240 Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys 245 250 255 Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr 260 265 270 Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe 275 280 285 Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu 290 295 300 Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala Gly Ile 305 310 315 320 Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe Gly Arg 325 330 335 Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr Ala Ser 340 345 350 Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His Asn Gly Met 355 360 365 Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu Gln Val 370 375 380 Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro Gln Ala Phe 385 390 395 400 Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His His Leu 405 410 415 Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His Ala Leu Val 420 425 430 Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala Asp Leu 435 440 445 Val Asp Gly Thr Met Glu Val Leu His His Leu Gly Ser Val Ala Gly 450 455 460 Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met 465 470 475 3847DNAArtificial sequencesynthetic oligonucleotide 38ccggaattcg gcgcgccgag ctcctcgagc aaatttacac attgcca 473947DNAArtificial sequencesynthetic oligonucleotide 39ccggaattcg gcgcgccgag ctcctcgagc aaatttacac attgcca 474047DNAArtificial sequencesynthetic oligonucleotide 40ccggaattcg gcgcgccgag ctcctcgagc aaatttacac attgcca 474148DNAArtificial sequencesynthetic oligonucleotide 41aaaactgcag gcggccgccc accgcggtgg gctggctatg aagaaatt 484227DNAArtificial sequencesynthetic oligonucleotide 42cgcggatccg ctggctatga agaaatt 274345DNAArtificial sequencesynthetic oligonucleotide 43tcccccggga tcgatgccgg cagatctgct ggctatgaag aaatt 454440DNAArtificial sequencesynthetic oligonucleotide 44aaaactgcag tctagaaggc ctcctgcttt aatgagatat 404551DNAArtificial sequencesynthetic oligonucleotide 45cgcggatccg atatcgggcc cgctagcgtt aaccctgctt taatgagata t 514633DNAArtificial sequencesynthetic oligonucleotide 46tcccccgggc catggcctgc tttaatgaga tat 334753DNAArtificial sequencesynthetic oligonucleotide 47cccaagcttg gcgcgccgag ctcgaattcg tcgacggaca atcagtaaat tga

534853DNAArtificial sequencesynthetic oligonucleotide 48cccaagcttg gcgcgccgag ctcgaattcg tcgacggaca atcagtaaat tga 534947DNAArtificial sequencesynthetic oligonucleotide 49cccaagcttg gcgcgccgag ctcgtcgacg gacaatcagt aaattga 475029DNAArtificial sequencesynthetic oligonucleotide 50acataatgga gaacttctgg tcgatcgtc 295124DNAArtificial sequencesynthetic oligonucleotide 51ttactcagat ttcttcccgt cttt 245226DNAArtificial sequencesynthetic oligonucleotide 52acataatgac cttcctagcc atatta 265324DNAArtificial sequencesynthetic oligonucleotide 53tcagatattc aaattggcgg cttc 245432DNAArtificial sequencesynthetic oligonucleotide 54ttaagcgcgg ccgcatggag aacttctggt cg 325531DNAArtificial sequencesynthetic oligonucleotide 55acctcggcgg ccgccctttt actcagattt c 315641DNAArtificial sequencesynthetic oligonucleotide 56acataatgga gaacttctgg tctattgttg tgttttttct a 415741DNAArtificial sequencesynthetic oligonucleotide 57ctagctagct tactcagatt tcttcccgtc ttttgtttct c 4158285PRTMus musculus 58Met Glu Leu Trp Pro Gly Ala Trp Thr Ala Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Ser Thr Leu Trp Phe Cys Ser Ser Ser Ala Lys Tyr Phe 20 25 30 Phe Lys Met Ala Phe Tyr Asn Gly Trp Ile Leu Phe Leu Ala Ile Leu 35 40 45 Ala Ile Pro Val Cys Ala Val Arg Gly Arg Asn Val Glu Asn Met Lys 50 55 60 Ile Leu Arg Leu Leu Leu Leu His Ala Lys Tyr Leu Tyr Gly Ile Arg 65 70 75 80 Val Glu Val Arg Gly Ala His His Phe Pro Pro Thr Gln Pro Tyr Val 85 90 95 Val Val Ser Asn His Gln Ser Ser Leu Asp Leu Leu Gly Met Met Glu 100 105 110 Val Leu Pro Asp Arg Cys Val Pro Ile Ala Lys Arg Glu Leu Leu Trp 115 120 125 Ala Gly Ser Ala Gly Leu Ala Cys Trp Leu Ala Gly Ile Ile Phe Ile 130 135 140 Asp Arg Lys Arg Thr Gly Asp Ala Ile Ser Val Met Ser Glu Val Ala 145 150 155 160 Gln Thr Leu Leu Thr Gln Asp Val Arg Val Trp Val Phe Pro Glu Gly 165 170 175 Thr Arg Asn His Asn Gly Ser Met Leu Pro Phe Lys Arg Gly Ala Phe 180 185 190 His Leu Ala Val Gln Ala Gln Val Pro Ile Ile Pro Ile Val Met Ser 195 200 205 Ser Tyr Gln Asp Phe Tyr Ser Lys Lys Glu Arg Arg Phe Thr Ser Pro 210 215 220 Gly Arg Cys Gln Val Arg Val Leu Pro Pro Val Ser Thr Glu Gly Leu 225 230 235 240 Thr Pro Asp Asp Val Pro Ala Leu Ala Asp Ser Val Arg His Ser Met 245 250 255 Leu Thr Ile Phe Arg Glu Ile Ser Thr Asp Gly Leu Gly Gly Gly Asp 260 265 270 Cys Leu Lys Lys Pro Gly Gly Ala Gly Glu Ala Arg Leu 275 280 285 59262PRTCaenorhabditis elegans 59Met Thr Phe Leu Ala Ile Leu Phe Val Ile Ala Val Leu Leu Leu Leu 1 5 10 15 Ala Gln Leu Pro Val Ile Gly Phe Tyr Ile Arg Ala Val Tyr Phe Gly 20 25 30 Met Cys Leu Ile Ile Gly Gly Phe Leu Gly Gly Leu Ala Ser Ile Pro 35 40 45 Phe Gly Lys Ser Pro Asn Asn His Phe Arg Met Phe Lys Ile Phe Gln 50 55 60 Ala Met Thr Trp Pro Met Gly Val Arg Phe Glu Leu Arg Asn Ser Glu 65 70 75 80 Ile Leu His Asp Lys Lys Pro Tyr Ile Ile Ile Ala Asn His Gln Ser 85 90 95 Ala Leu Asp Val Leu Gly Met Ser Phe Ala Trp Pro Val Asp Cys Val 100 105 110 Val Met Leu Lys Ser Ser Leu Lys Tyr Leu Pro Gly Phe Asn Leu Cys 115 120 125 Ala Tyr Leu Cys Asp Ser Val Tyr Ile Asn Arg Phe Ser Lys Glu Lys 130 135 140 Ala Leu Lys Thr Val Asp Thr Thr Leu His Glu Ile Val Thr Lys Lys 145 150 155 160 Arg Lys Val Trp Ile Tyr Pro Glu Gly Thr Arg Asn Ala Glu Pro Glu 165 170 175 Leu Leu Pro Phe Lys Lys Gly Ala Phe Ile Leu Ala Lys Gln Ala Lys 180 185 190 Ile Pro Ile Val Pro Cys Val Phe Ser Ser His Lys Phe Phe Tyr Ser 195 200 205 His Ala Glu Lys Arg Leu Thr Ser Gly Asn Cys Ile Ile Asp Ile Leu 210 215 220 Pro Glu Val Asp Ser Ser Lys Phe Asp Ser Ile Asp Asp Leu Ser Ala 225 230 235 240 His Cys Arg Lys Ile Met Gln Ala His Arg Glu Lys Leu Asp Ala Glu 245 250 255 Ala Ala Asn Leu Asn Ile 260

Claims

1. A transgenic plant or plant cell comprising a heterologous acyl-CoA:lysophospholipid-acyltransferase (LPLAT) gene, wherein said plant or plant cell has an increased content of polyunsaturated fatty acids in comparison with a corresponding non-transgenic plant or plant cell lacking said heterologous LPLAT gene.

2. The transgenic plant or plant cell of claim 1, wherein said heterologous LPLAT gene is integrated into the genome of said plant or plant cell and replaces a LPLAT gene native to said plant or plant cell by recombination.

3. The transgenic plant or plant cell of claim 1, wherein the polyunsaturated fatty acids produced in said plant or plant cell comprises C₁₈-, C₂₀- and/or C₂₂-fatty acids with at least two double bonds.

4. The transgenic plant or plant cell of claim 1, further comprises at least one heterologous nucleic acid encoding an enzyme of the fatty acid or lipid metabolism.

5. The transgenic plant or plant cell of claim 4, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases and fatty acid elongase(s).

6. The transgenic plant or plant cell of claim 4, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of Δ4-desaturases, Δ5-desaturases, Δ6-desaturases, Δ8-desaturases, Δ9-desaturases, Δ12-desaturases, Δ5-elongases, Δ6-elongases and Δ9-elongases.

7. The transgenic plant or plant cell of claim 5, wherein the plant is an oil crop plant, or wherein the plant cell is an oil crop plant cell.

8. A method for modulating production of polyunsaturated fatty acids in a non-human organism, comprising: (a) obtaining a non-human organism comprising a heterologous LPLAT gene; and (b) growing said non-human organism under conditions so that said heterologous LPLAT gene is expressed, wherein expression of said heterologous LPLAT gene modifies production of polyunsaturated fatty acids in said non-human organism.

9. The method of claim 8, wherein said heterologous LPLAT gene is integrated into the genome of said non-human organism and replaces a LPLAT gene native to said non-human organism by recombination.

10. The method of claim 8, wherein the polyunsaturated fatty acids produced in said non-human organism comprises C₁₈-, C₂₀- and/or C₂₂-fatty acids with at least two double bonds.

11. The method of claim 8, wherein the non-human organism further comprises at least one heterologous nucleic acid encoding an enzyme of the fatty acid or lipid metabolism.

12. The method of claim 11, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases and fatty acid elongase(s).

13. The method of claim 11, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of Δ4-desaturases, Δ5-desaturases, Δ6-desaturases, Δ8-desaturases, Δ9-desaturases, Δ12-desaturases, Δ5-elongases, Δ6-elongases and Δ9-elongases.

14. The method of claim 8, wherein the non-human organism is an oil crop plant.

15. A transgenic plant or plant cell capable of producing docosahexaenoic acid (DHA), wherein said plant or plant cell comprises a heterologous LPLAT gene, and wherein expression of said heterologous LPLAT gene leads to the production of DHA in said plant or plant cell.

16. The transgenic plant or plant cell of claim 15, wherein said heterologous LPLAT gene is integrated into the genome of said plant or plant cell and replaces a LPLAT gene native to said plant or plant cell by recombination.

17. The transgenic plant or plant cell of claim 15, further comprises at least one heterologous nucleic acid encoding an enzyme of the fatty acid or lipid metabolism.

18. The transgenic plant or plant cell of claim 17, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyltransferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allenoxide synthases, hydroperoxide lyases and fatty acid elongase(s).

19. The transgenic plant or plant cell of claim 17, wherein the at least one heterologous nucleic acid encodes an enzyme of the fatty acid or lipid metabolism selected from the group consisting of Δ4-desaturases, Δ5-desaturases, Δ6-desaturases, Δ8-desaturases, Δ9-desaturases, Δ12-desaturases, Δ5-elongases, Δ6-elongases and Δ9-elongases.

20. The transgenic plant or plant cell of claim 15, wherein the plant is an oil crop plant, or wherein the plant cell is an oil crop plant cell.