Patent application title: Feedstuffs for Aquaculture Comprising Stearidonic Acid
Inventors:
Matthew Robert Miller (Atawhai, NZ)
Christopher Guy Carter (Howden, AU)
Peter David Nichols (West Hobart, AU)
Peter David Nichols (West Hobart, AU)
Surinder Pal Singh (Downer, AU)
Surinder Pal Singh (Downer, AU)
Xue-Rong Zhou (Evatt, AU)
Xue-Rong Zhou (Evatt, AU)
Allan Graham Green (Red Hill, AU)
Allan Graham Green (Red Hill, AU)
Assignees:
COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION
IPC8 Class: AA23K118FI
USPC Class:
119230
Class name: Aquatic animal culturing fish culturing feeding method
Publication date: 2014-11-27
Patent application number: 20140345535
Abstract:
The present invention relates to feedstuffs for use in aquaculture, as
well as methods for producing said feedstuffs. The invention also
provides methods for rearing fish and/or crustaceans. In particular, the
present invention provides a method of rearing a fish or crustacean, the
method comprising feeding the fish or crustacean a feedstuff comprising
lipid, the fatty acid of said lipid comprising at least 5.5% (w/w)
stearidonic acid (SDA).Claims:
1-60. (canceled)
61. A process for increasing the long-chain polyunsaturated fatty acid (LC-PUFA) content in the white muscle, the red muscle, or the white and red muscle of a fish comprising feeding the fish a feedstuff comprising plant oil whose fatty acids comprise at least 5.5% (w/w) stearidonic acid (SDA), so as to thereby increase the LC-PUFA content of the white muscle, the red muscle, or the white and red muscle of the fish relative to the white muscle, the red muscle, or the white and red muscle of a corresponding fish fed a feedstuff comprising canola oil and whose fatty acids lack at least 5.5% (w/w) SDA, and wherein levels of 14:0 and 16:0 fatty acids in muscle tissue of the fish are reduced by at least 10% relative to a corresponding fish fed a corresponding feedstuff which comprises fish oil instead of the plant oil whose fatty acids comprise at least 5.5% (w/w) SDA.
62. The process of claim 61, wherein the LC-PUFA is DHA.
63. The process of claim 62, wherein the DHA content of the fatty acids of the white muscle lipid of the fish is increased to comprise 18.3% (w/w) DHA.
64. The process of claim 62, wherein the DHA content of the fatty acids of the red muscle lipid of the fish is increased to comprise 9.6% (w/w) DHA.
65. The process of claim 61, wherein the fish is a trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod, haddock, Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike, pike-perch, yellowtail, tilapia, eel or tropical fish.
66. The process of claim 61, wherein the fish is a larval or a juvenile fish.
67. The process of claim 61, wherein the fish is fed the feedstuff for at least 6 weeks.
68. The process of claim 61, wherein the plant oil is from a plant which is canola, soybean, or flax.
69. The process of claim 68, wherein the plant is genetically modified such that the fatty acids of the oil comprise at least 5.5% (w/w) SDA.
70. The process of claim 69, wherein the plant comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
71. The process of claim 61, wherein the feedstuff comprises a transgenic organism, or extract or portion thereof and at least one other ingredient, wherein the organism is genetically modified such that the fatty acids of the organism, or extract or portion thereof comprise at least 5.5% (w/w) SDA.
72. The process of claim 71, wherein the organism is yeast.
73. The process of claim 71, wherein the transgenic organism, or extract or portion thereof comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
74. The process of claim 65, wherein the feedstuff comprises plant oil and at least one other ingredient, wherein the fatty acids of the oil comprise at least 5.5% (w/w) SDA.
75. The process of claim 74, wherein the plant oil is from a plant which is canola, soybean, or flax.
76. The process of claim 65, wherein the feedstuff comprises a transgenic organism, or extract or portion thereof and at least one other ingredient, wherein the organism is genetically modified such that the fatty acids of the organism, or extract or portion thereof comprise at least 5.5% (w/w) SDA.
77. The process of 76, wherein the organism is yeast.
78. The process of claim 76, wherein the transgenic organism, or extract or portion thereof comprises a polypeptide having an amino acid sequence at least 85% identical to the amino acid sequence of the protein encoded by the nucleotide sequence set forth in Genbank Accession No. AY234127, and having Δ6 desaturase activity.
79. The process of claim 76, wherein the at least one other ingredient is fishmeal, a starch source, a vitamin or a mineral.
Description:
[0001] This application claims priority from U.S. 60/737,946, the entire
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to feedstuffs for use in aquaculture, as well as methods for producing said feedstuffs. The invention also provides methods for rearing fish and/or crustaceans.
BACKGROUND OF THE INVENTION
[0003] Global production of farmed fish and crustacea has more than doubled in the last 15 years and its expansion places an increasing demand on global supplies of wild fish harvested to provide protein and oil as ingredients for aquafeeds (Naylor et al., 2000). The supply of seafood from global capture fisheries sources is around 100 million tones per annum (FAO, 2001). This amount has not increased since the mid-1980's and will not increase in the future as most fisheries are at or above sustainable levels of production, and are further subjected to sharp, periodic declines, due to climatic factors such as El Nino (FAO, 2001; Barlow 2000). Fish oil stocks are also under increasing demand not only from aquaculture, but from the agriculture and nutraceutical/biomedical industries.
[0004] Replacement oils for the aquaculture industry have been sourced from a variety of commercial terrestrial plant sources including sunflower (Bransden et al, 2003; Bell et al., 1993), canola/rapeseed (Bell et al, 2003; Polvi and Ackman, 1992), olive, palm (Fonseca-Madrigal et al, 2005; Bell et al, 2002) and linseed (Bell et al., 1993; Bell et al., 2004). The inclusion of vegetable oil to replace part or all of the fish oil in fish diets resulted in the same growth rates and feed conversion ratios (Bransden et al., 2003; Polvi and Ackman, 1992; Torstensen et al., 2004; Fonseca-Magrigal et al., 2005; Bell et al., 2002; Bell et al., 2004). However, since these plant oils had essentially no ω3 long-chain (≧C20) polyunsaturated fatty acids (ω3 LC-PUFA) and had high levels of monounsaturated fatty acids (MUFA), ω6 PUFA and low ω3/ω6 ratios, fish fed such diets displayed reduced levels of ω3 LC-PUFA. This is thought to be associated with reduced health benefits to the consumer compared to fish fed a diet high in fish oil containing greater levels of ω3 LC-PUFA (Seierstad et al., 2005). Therefore, raising fish or crustacea on diets high in vegetable oil has the potential to dilute the important cardiovascular and other benefits which are associated with eating fish.
Pathways of LC-PUFA Synthesis
[0005] Biosynthesis of LC-PUFA from linoleic and α-linolenic fatty acids in organisms such as microalgae, mosses and fungi may occur by a series of alternating oxygen-dependent desaturations and elongation reactions as shown schematically in FIG. 1. In one pathway (FIG. 1, II), the desaturation reactions are catalysed by Δ6, Δ5, and Δ4 desaturases, each of which adds an additional double bond into the fatty acid carbon chain, while each of a Δ6 and a Δ5 elongase reaction adds a two-carbon unit to lengthen the chain. The conversion of ALA to DHA in these organisms therefore requires three desaturations and two elongations. Genes encoding the enzymes required for the production of DHA in this aerobic pathway have been cloned from various microorganisms and lower plants including microalgae, mosses, fungi.
[0006] Alternative routes have been shown to exist for two sections of the ALA to DHA pathway in some groups of organisms. The conversion of ALA to ETA may be carried out by a combination of a Δ9 elongase and a Δ8 desaturase (the so-called Δ8 desaturation route, see FIG. 1, IV) in certain protists and thraustochytrids, as evidenced by the isolated of genes encoding such enzymes (Wallis and Browse, 1999; Qi et al., 2002). In mammals, the so-called "Sprecher" pathway converts DPA to DHA by three reactions, independent of a Δ4 desaturase (Sprecher et al., 1995).
[0007] Besides these desaturase/elongase systems, EPA and DHA can also be synthesized through an anaerobic pathway in a number of organisms such as Shewanella, Mortiella and Schizochytrium (Abbadi et al., 2001). The operons encoding these polyketide synthase (PKS) enzyme complexes have been cloned from some bacteria (Morita et al., 2000; Metz et al., 2001; Tanaka et al., 1999; Yazawa, 1996; Yu et al., 2000; WO 00/42195). The EPA PKS operon isolated from Shewanella spp has been expressed in Synechococcus allowing it to synthesize EPA (Takeyama et al., 1997). The genes encoding these enzymes are arranged in relatively large operons, and their expression in transgenic plants has not been reported. Therefore it remains to be seen if the anaerobic PKS-like system is a possible alternative to the more classic aerobic desaturase/elongase for the transgenic synthesis of LC-PUFA.
[0008] The biosynthetic pathways for PUFA are well known (Sargent et al., 2002). Vertebrates lack ω12 and ω15 (ω3) lipid desaturases and cannot produce linoleic acid (18:2ω6, LA) and α-linolenic acid (18:ω3, ALA) from oleic acid (18:ω9, OA) (see FIG. 1). The conversion from ALA to eicosapentaenoic acid (20:5ω3, EPA) and docosahexaenoic acid (22:6ω3, DHA) is inefficient in marine fish, which have high levels of LC-PUFA in their natural diet, but is greater in freshwater fish, which have high levels of LA and ALA and limited DHA in their natural diet. High levels of ω3 LC-PUFA, which are found in salmon, cannot be biosynthesised from ALA and LA and therefore must be provided to the fish in their diet.
Desaturases
[0009] The desaturase enzymes that have been shown to participate in LC-PUFA biosynthesis all belong to the group of so-called "front-end" desaturases which are characterised by the presence of a cytochrome b5 domain at the N-terminus of each protein. The cyt b5 domain presumably acts as a receptor of electrons required for desaturation (Sperling and Heinz, 2001). The enzyme Δ6 desaturase catalyses the desaturation of linoleic acid (LA) to form gamma-linoleic acid (GLA, 18:3ω6) and linolenic acid (ALA) to form stearidonic acid (SDA, 18:4ω3) (FIG. 1). Genes encoding this enzyme have been isolated from a number of organisms, including plants, mammals, nematodes, fungi and marine microalgae. The C18 fatty acid substrate for Δ6 desaturases from plants, fungi and microalgae has desaturation in at least the Δ9 and Δ12 positions and is generally covalently linked to a phosphatidylcholine headgroup (acyl-PC).
[0010] The enzyme Δ5 desaturase catalyses the desaturation of C20 LC-PUFA leading to arachidonic acid (ARA, 20:4 ω6) and EPA (20:5ω3). Genes encoding this enzyme have been isolated from a number of organisms, including algae (Thraustochytrium sp. Qiu et al., 2001), fungi (M. alpine, Pythium irregulare, Michaelson et al., 1998; Hong et al., 2002), Caenorhabditis elegans and mammals. A gene encoding a bifunctional Δ5-/Δ6-desaturase has also been identified from zebrafish (Hasting et al., 2001). The gene encoding this enzyme might represent an ancestral form of the "front-end desaturase" which later duplicated and evolved distinct functions.
[0011] The last desaturation step to produce DHA is catalysed by a Δ4 desaturase and a gene encoding this enzyme has been isolated from the freshwater protist species Euglena gracilis and the marine species Thraustochytrium sp. (Qiu et al., 2001; Meyer et al., 2003).
Elongases
[0012] Several genes encoding PUFA-elongation enzymes have also been isolated (Sayanova and Napier, 2004). The members of this gene family were unrelated to the elongase genes present in higher plants, such as FAE1 of Arabidopsis, that are involved in the extension of saturated and monounsaturated fatty acids. An example of the latter is erucic acid (22:1) in Brassicas. In some protist species, LC-PUFA are synthesized by elongation of linoleic or α-linolenic acid with a C2 unit, before desaturation with Δ8 desaturase (FIG. 1 part IV; "Δ8-desaturation" pathway). Δ6 desaturase and Δ6 elongase activities were not detected in these species. Instead, a Δ9-elongase activity would be expected in such organisms, and in support of this, a C18 Δ9-elongase gene has recently been isolated from Isochrysis galbana (Qi et al., 2002).
Transgenic Plants
[0013] Transgenic oilseed crops that are engineered to produce major LC-PUFA by the insertion of various genes encoding desaturases and/or elongases have been suggested as a sustainable source of nutritionally important fatty acids. However, the requirement for coordinate expression and activity of five new enzymes encoded by genes from possibly diverse sources has made this goal difficult to achieve and only low yields have generally been obtained (reviewed by Sayanova and Napier, 2004; Drexler et al., 2003; Abbadi et al., 2001).
[0014] A gene encoding a Δ6-fatty acid desaturase isolated from borage (Borago officinalis) was expressed in transgenic tobacco and Arabidopsis, resulting in the production of GLA (18:3ω6) and SDA (18:4ω3), the direct precursors for LC-PUFA, in the transgenic plants (Sayanova et al., 1997 and 1999). However, this provides only a single, first step.
Feedstuffs for Aquaculture
[0015] Research in feedstuffs for aquaculture have largely focused on enriching salmon diets by increasing the dietary supply of ALA (Bell et al., 1993) and EPA/DHA (Harel et al., 2002; Carter et al., 2003).
[0016] There is a need for further diets for aquaculture which, upon consumption, enhance the production of omega-3 long chain polyunsaturated fatty acids in aquatic animals.
SUMMARY OF THE INVENTION
[0017] The present inventors have determined that fish and crustaceans can be produced with appropriate levels of LC-PUFA, such as EPA, DPA and/or DHA, without the need to feed these organisms diets which are rich in LC-PUFA. In particular, the LC-PUFA precursor stearidonic acid (SDA) can be provided to the fish or crustaceans whilst still producing fish or crustaceans with desirable levels of LC-PUFA.
[0018] Thus, in a first aspect, the present invention provides a method of rearing a fish or crustacean, the method comprising feeding the fish or crustacean a feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA).
[0019] In a preferred embodiment, the lipid comprises a phytosterol.
[0020] In a particularly preferred embodiment, at least 1% of the SDA in the feedstuff was obtained from a plant. The plant may be non-transgenic, such as an Echium sp., Oenothera biennis, Borago officinalis or Ribes nigrum, or transgenic. In an embodiment, at least some of the SDA is from oil obtained from seed of the plant.
[0021] In a preferred embodiment, the transgenic plant comprises an exogenous nucleic acid encoding a Δ6 desaturase. The transgenic plant may further comprise an exogenous nucleic acid encoding a ω3 desaturase or Δ15 desaturase, which increases the production of ALA in the plant. The transgenic plant may further comprise an exogenous nucleic acid encoding a Δ12 desaturase. Examples of suitable transgenic plants include, but are not limited to, canola, soybean, flax, other oilseed plants, cereals or grain legumes.
[0022] In a particularly preferred embodiment, the fish is a salmon.
[0023] In one embodiment, the fish or crustacean is fed predominantly the feedstuff over a period of at least 6 weeks, preferably at least 7 weeks and even more preferably at least 12 weeks. In an embodiment, after having been fed the feedstuff for at least 6 weeks, the fish or crustacean has similar weight, specific growth rate, weight gain, total feed consumption, feed efficiency ratio, hepatosomatic index and/or survival when compared with the same species of fish or crustacean fed the same feedstuff but which substantially lacks SDA.
[0024] In another embodiment, the fish or crustacean, after having been fed the feedstuff for at least 6 weeks, has higher SDA and/or ETA levels in muscle tissue when compared with the same species of fish or crustacean fed the same feedstuff but which substantially lacks SDA.
[0025] In a further embodiment, the fish or crustacean, after having been fed the feedstuff for at least 6 weeks, has lower SFA levels in muscle tissue when compared with the same species of fish or crustacean fed the same feedstuff but which comprises fish oil instead of the plant oil comprising at least 5.5% SDA. In preferred embodiments, the levels of 14:0 and 16:0 are reduced, for example by at least 10% or at least 20%.
[0026] In another aspect, the present invention provides a feedstuff for a fish or crustacean, the feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA, 18:4Δ6,9,12,15, ω33). The feedstuff may have any of the characteristics as described herein in the context of the methods.
[0027] In a further aspect, the present invention provides a fish or crustacean produced using a method of the invention.
[0028] In yet another aspect, the present invention provides a fish, wherein the fatty acid of the white muscle lipid of said fish comprises less than 29.6% SFA and at least 18.3% DHA. In certain embodiments, the white muscle lipid of the fish comprises fatty acid comprising less than 28%, less than 27%, or more preferably less than 26% SFA. In other embodiments, the white muscle lipid of the fish comprises fatty acid comprising at least 19%, at least 20%, at least 21%, or more preferably at least 22% DHA.
[0029] In another aspect, the present invention provides a fish, wherein the fatty acid of the red muscle lipid of said fish comprises fatty acid comprising less than 28.2% SFA and at least 9.6% DHA. In certain embodiments, the red muscle lipid of the fish comprises fatty acid comprising less than 27%, less than 26%, or more preferably less than 25% SFA. In other embodiments, the muscle lipid of the fish comprises fatty acid comprising at least 10%, at least 11%, or more preferably at least 12% DHA.
[0030] In a further aspect, the present invention provides a fish or crustacean, wherein the fatty acid of the muscle lipid of said fish or crustacean comprises at least 2.7% SDA. In embodiments of this aspect, the muscle lipid of said fish or crustacean comprises at least 3%, at least 3.5%, or more preferably at least 4% SDA.
[0031] In a further aspect, the present invention provides a fish, wherein the fatty acid of the white muscle lipid of said fish comprises at least 2.1% SDA. In embodiments of this aspect, the white muscle lipid of said fish comprises at least 2.5%, at least 3%, or more preferably at least 3.5% SDA.
[0032] Preferably, a fish of the invention is a salmon.
[0033] In yet a further aspect, the present invention provides a method for producing a feedstuff for fish and/or crustaceans, the method comprising admixing oil obtained from a plant with at least one other ingredient, wherein the fatty acid of said oil comprises at least 5.5% (w/w) SDA. In a preferred embodiment, the other ingredient comprises fish meal, a high protein source other than fishmeal, a starch source or a combination of these. Other ingredients may include vitamins, minerals, choline, or pigments such as, for example, carotenoids or carophyll pink.
[0034] Preferably, the plant is transgenic.
[0035] Preferably, the oil is obtained from the seed of the plant.
[0036] In certain embodiments, it is preferred that the fatty acid of said oil comprises at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11.0%, at least 15%, at least 20%, or at least 30% (w/w) SDA.
[0037] In another aspect, the present invention provides a method for producing a feedstuff for fish and/or crustaceans, the method comprising admixing a transgenic organism, or extract or portion thereof, with at least one other ingredient, wherein the organism is genetically modified such that it produces SDA and/or produces higher levels of SDA than when compared to a corresponding non-transgenic wild-type organism. The method may comprise the step of extracting the oil from the organism, for example from the seed of a plant. The extraction may comprise physical means such as crushing of seed, chemical means such as extraction with a solvent, heating or other processes, or any combination of these. The oil may be further purified before mixing with other ingredients. The method preferably includes preparation of an extruded product from the mixed ingredients by an extrusion process, suitable for providing to fish or crustacean. The method may comprise the step of analysing the feedstuff, such as for example measuring the level of lipid or the level of SDA in the fatty acid, or other measurements.
[0038] Preferably, the organism is a plant or yeast.
[0039] In another aspect, the present invention provides a feedstuff produced using a method of the invention. The feedstuff may have the characteristics as described above. Other ingredients that may be included in the feedstuff include fish meal, a high protein source other than fishmeal, a starch source, vitamins, minerals, pigments such as, for example, carotenoids or carophyll pink, or any combination of these. Fishmeal is a preferred protein source for the major carnivorous fish such as salmon, trout, tuna, flatfish, barramundi, particularly for Atlantic salmon. Fishmeal, typically about 65% protein, may be added in an amount from 20 to 700 g per kg dryweight. A high protein source other than fishmeal may be from a plant or animal source such as, for example, wheat or other cereal gluten, soymeal, meal from other legumes, casein, protein concentrates, protein isolates, meat, meat and bone, blood, feathers. These are typically at least 30% protein and may be milled with or without extraction of oil. Starch may be added, typically at 10-150 g/kg, and may be in the form of cereal grain or meal. For crustaceans, krill meal, mussel meal or other similar components may be added at 1-200 g/kg, cholesterol and/or lecithin at 0-100 g/kg. The mixture may comprise a binding agent such as sodium alginate, for example Manucol from Kelco International.
[0040] In a further aspect, the present invention provides oil extracted from a fish or crustacean of the invention, comprising SDA, EPA, DPA, DHA or any combinations thereof.
[0041] In yet another aspect, the present invention provides a cotton or flax plant capable of producing seed, wherein the oil of said seed comprises fatty acid comprising at least 5.5% SDA on a weight basis.
[0042] Furthermore, the present inventors have found that expressing a Δ6 desaturase gene in a fibre producing plant results in surprisingly high levels of Δ6 desaturase PUFA products.
[0043] Thus, in a further aspect the present invention provides a cotton or flax plant capable of producing seed, wherein the seed synthesizes GLA that is the product of Δ6-desaturation of LA and/or SDA that is the product of Δ6-desaturation of ALA, and wherein the efficiency of conversion of LA to GLA and/or ALA to SDA in the seed is at least 25%, at least 35%, or at least 45%. For example, at least 25%, preferably at least 45% of the polyunsaturated fatty acid in the cotton or flax seed that has a carbon chain of C18 or longer is desaturated at the Δ6 position.
[0044] Preferably, the cotton plant is Gossypium hirstum or Gossypium barbadense.
[0045] Preferably, the flax plant is Linum usitatissimum.
[0046] Preferably, the fatty acid of the oil comprises at least 8% SDA, or at least 10% SDA, at least 11% SDA, at least 15% SDA, at least 20% SDA, at least 25% SDA, at least 30% SDA, at least 35% SDA, at least 40% SDA, at least 45% SDA or at least 50% SDA.
[0047] In one preferred embodiment, the plant comprises a transgenic Δ6 desaturase gene. In another preferred embodiment, the plant comprises a transgenic Δ15 desaturase or ω3 desaturase gene which may be in additional to the transgenic Δ6 desaturase gene. In an embodiment, the protein coding region of said gene is from a plant, microalgal, fungal or vertebrate source.
[0048] Also provided is the seed of a plant of the invention, wherein the oil of said seed comprises fatty acid comprising at least 5.5% SDA on a weight basis.
[0049] In a further aspect, the present invention provides a method of producing a plant of the invention, comprising the introduction of a Δ6 desaturase gene into a cotton or flax plant cell and the regeneration of a plant therefrom.
[0050] In an embodiment, the method comprises the step of determining the fatty acid composition of seedoil obtained from seed of said plant and/or the step of selecting a plant on the basis of its seed oil composition.
[0051] In another embodiment, the method further comprises the introduction of a Δ15 desaturase or ω3 desaturase gene into said plant.
[0052] In yet a further aspect, the present invention provides a method of producing the seed of the invention, comprising growing said plant and harvesting seed from said plant.
[0053] As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
[0054] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
[0055] The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0056] FIG. 1. Possible pathways of ω3 and ω6 LC-PUFA synthesis. The sectors labelled I, II, III, and IV correspond to the ω6 (Δ6), ω3 (Δ6), ω6 (Δ8), and ω3 (Δ8) pathways, respectively. Compounds in sectors I and III are ω6 compounds, while those in sectors II and IV are ω3 compounds. "Des" refers to desaturase steps in the pathway catalysed by desaturases as indicated, while "Elo" refers to elongase steps catalysed by elongases as indicated. The thickened arrow indicates the Δ5 elongase step. The dashed arrows indicate the steps in the "Sprecher" pathway that operates in mammalian cells for the production of DHA from DPA.
[0057] FIG. 2. Schematic representation of the construct, pVLin-Ed6, used to transform flax. RB, right border of T-DNA; HPT+Cat-1, hygromycin resistance gene interrupted by Cat-1 intron; 35SP, Cauliflower mosaic virus 35S promoter; LinT, Linin terminator; ED6, full length coding sequence of Δ6 fatty acid desaturase from Echium; LinP, linin promoter; LB, left border of T-DNA. P, PstI; A, ApaI; X, XhoI; N, NotI.
KEY TO THE SEQUENCE LISTING
[0058] SEQ ID NO:1--Δ6 desaturase from humans (Genbank Accession No: AAD20018). SEQ ID NO:2--Δ6 desaturase from mouse (Genbank Accession No: NP--62673). SEQ ID NO:3--Δ6 desaturase from Pythium irregulare (Genbank Accession No: AAL13310). SEQ ID NO:4--Δ6 desaturase from Borago officinalis (Genbank Accession No: AAD01410). SEQ ID NO:5--Δ6 desaturase from Anemone leveillei (Genbank Accession No: AAQ10731). SEQ ID NO:6--Δ6 desaturase from Ceratodon purpureus (Genbank Accession No: CAB94993). SEQ ID NO:7--Δ6 desaturase from Physcomitrella patens (Genbank Accession No: CAA11033). SEQ ID NO:8--Δ6 desaturase from Mortierella alpina (Genbank Accession No: BAC82361). SEQ ID NO:9--Δ6 desaturase from Caenorhabditis elegans (Genbank Accession No: AAC15586). SEQ ID NO:10--Δ6 desaturase from Echium plantagineum. SEQ ID NO:11--Δ6 desaturase from Echium gentianoides (Genbank Accession No: AY055117). SEQ ID NO:12--Δ6 desaturase from Echium pitardii (Genbank Accession No: AY055118). SEQ ID NO:13--Δ5/Δ6 bifunctional desaturase from Danio rerio (zebrafish). SEQ ID NO's 14 to 16--Conserved motifs of Echium sp. Δ6 desaturases. SEQ ID NO's 17 to 22, 30 and 31--Oligonucleotide primers. SEQ ID NO:23--Linin promoter from Linum usitatissimum. SEQ ID NO:24--Linin terminator from Linum usitatissimum. SEQ ID NO:25--cDNA sequence encoding Δ6 desaturase from Echium plantagineum. SEQ ID NO:26--Δ15 desaturase from Perilla frutescens (Genbank Accession No: AF213482). SEQ ID NO:27--Δ15 desaturase from Brassica napus (Genbank Accession No: L01418). SEQ ID NO:28--Δ15 desaturase from Betula pendula (Genbank Accession No: AAN17504). SEQ ID NO:29--Δ15 desaturase from Arabidposis thaliana (Genbank Accession No: AAC31854).
DETAILED DESCRIPTION OF THE INVENTION
General Techniques and Definitions
[0059] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, plant biology, molecular genetics, immunology, immunohistochemistry, fatty acid synthesis, protein chemistry, and biochemistry).
[0060] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and are incorporated herein by reference.
[0061] As used herein, the term "lipid" generally refers to an organic molecule, typically containing a hydrocarbon chain(s), that is insoluble in water but dissolves readily in nonpolar organic solvents. Feedstuffs of the invention are defined herein relative to the composition of their lipid component. This lipid component includes fatty acids (either free or esterified, for example in the form of triacylglycerols), sterols and polar lipids.
[0062] As used herein, the term "fatty acids" refers to a large group of organic acids made up of molecules containing a carboxyl group at the end of a hydrocarbon chain; the carbon content may vary from C2 to C34. The fatty acids may be saturated (contain no double bonds in the carbon chain) (SFA), monounsaturated (contain a single double bond in the carbon chain) (MUFA), or polyunsaturated (contain a two, three, four or more double bonds in the carbon chain) (PUFA). Unless stated to the contrary, the fatty acids may be in a free state (non-esterified) or in an esterified form such as part of a triacylglycerol, diacylglyceride, monoacylglyceride, acyl-CoA bound or other bound form, or mixture thereof. The fatty acid may be esterified as a phospholipid such as a phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms.
[0063] As used herein, the terms "long-chain polyunsaturated fatty acid", "LC-PUFA" or "C20+ polyunsaturated fatty acid" refer to a fatty acid which comprises at least 20 carbon atoms in its carbon chain and at least three carbon-carbon double bonds. Ordinarily, the number of carbon atoms in the carbon chain of the fatty acids refers to an unbranched carbon chain. If the carbon chain is branched, the number of carbon atoms excludes those in side groups. Generally, the long-chain polyunsaturated fatty acid is an ω3 fatty acid, that is, having a desaturation (carbon-carbon double bond) in the third carbon-carbon bond from the methyl end of the fatty acid. Preferably, the long-chain polyunsaturated fatty acid is selected from the group consisting of; eicosatetraenoic acid (ETA, 20:4Δ8,11,14,17, ω3) eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17; ω3), docosapentaenoic acid (DPA, 22:5Δ7,10,13,16,19, ω3), or docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19, ω3). It would readily be apparent that the LC-PUFA that is in (or limited in amount or even excluded from) a feedstuff of the invention, or produced by a fish or crustacean fed a feedstuff of the invention, may be a mixture of any or all of the above and may include other LC-PUFA or derivatives of any of these LC-PUFA.
[0064] Use of the term "fish" includes all vertebrate fish, which may be bony or cartilaginous fish. The present invention may be practiced with any of the considerable variety of fresh, brackish, or salt water fish species including, but not limited to, salmon, trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, flounder, sea bass, cod, haddock, Japanese flounder, catfish, char, whitefish, sturgeon, tench, roach, pike, pike-perch, yellowtail, tilapia, eel or tropical fish (such as the fresh, brackish, and salt water tropical fish). In an embodiment, the fish is not hybrid striped bass. In a further embodiment, if the fish is hybrid striped bass, the fatty acid of said lipid comprises at least 11.0%, at least 12% or at least 15% (w/w) SDA. In another embodiment, if the fish is hybrid striped bass, the SDA content of the feedstuff is at least 2.1% (w/w). Yet other species with which the present invention can be practiced will be apparent to those skilled in the art, including those species outlined in Table 1. The invention may be practised with any, all, or any combination of the listed fish.
TABLE-US-00001 TABLE 1 Fish that can be fed feedstuffs of the invention. Family Scientific name Common name ACIPENSERIDAE Acipenser baeri Siberian sturgeon Acipenser ruthenus Sterlet sturgeon Acipenser stellatus Starry sturgeon Acipenser transmontanus White sturgeon Huso huso Beluga OSTEOGLOSSIDAE Arapaima gigas Arapaima Anguilla japonica Japanese eel Anguilla rostrata American eel Anguilla australis Short-finned eel Anguilla reinhardtii Long-finned eel Anguilla anguilla European eel CHANIDAE Chanos chanos Milkfish CYPRINIDAE Abramis brama Freshwater bream Aspius aspius Asp Catla catla Catla Carassius auratus Goldfish Carassius carassius Crucian carp Cirrhinus molitorella Mud carp Cirrhinus mrigala Mrigal carp Ctenopharyngodon idellus Grass carp Cyprinus carpio Common carp Hypophthalmichthys Silver carp molitrix Hypophthalmichthys nobilis Bighead carp Labeo calbasu Orangefin labeo Labeo rohita Roho labeo Leptobarbus hoeveni Hoven's carp Megalobrama Wuchang bream amblycephala Mylopharyngodon piceus Black carp Notemigonus crysoleucas Golden shiner Osteochilus hasselti Nilem carp Parabramis pekinensis White amur bream Puntius gonionotus Thai silver barb Puntius javanicus Java Rutilus rutilus Roach Tinca tinca Tench COBITIDAE Misgurnus anguillicaudatus Pond loach CURIMATIDAE Ichthyoelephas humeralis Bocachico Bocachico Prochilodus reticulatus Bocachico CHARACIDAE Brycon moorei Dorada Colossoma macropomum Cachama Piaractus brachypomus Cachama blanca Piaractus mesopotamicus Paco ICTALURIDAE Ictalurus melas Black bullhead Ictalurus punctatus Channel catfish BAGRIDAE Chrysichthys nigrodigitatus Bagrid catfish SILURIDAE Siluris glanis Wels catfish PANGASIIDAE Pangasius pangasius Pangas catfish Pangasius sutchi Striped catfish CLARIIDAE Clarias anguillaris Mudfish Clarias batrachus Philippine catfish Clarias fuscus Hong Kong catfish Clarias gariepinus North African catfish Clarias macrocephalus Bighead catfish Heterobranchus bidorsalis African catfish Heterobranchus longifilis Sampa PIMELODIDAE Rhamdia sapo South American catfish CALLICHTHYIDAE Hoplosternum littorale Atipa ESOCIDAE Esox lucius Northern pike PLECOGLOSSIDAE Plecoglossus altivelis Ayu sweetfish SALMONIDAE Coregonus albula Vendace Coregonus lavaretus Whitefish Oncorhynchus gorbuscha Pink salmon Oncorhynchus keta Chum salmon Oncorhynchus kisutch Coho salmon Oncorhynchus masou Masu salmon Oncorhynchus mykiss Rainbow trout Oncorhynchus nerka Sockeye salmon Oncorhynchus tshawytscha Chinook salmon Salmo salar Atlantic salmon Salmo trutta Sea trout Salvelinus alpinus Arctic char Salvelinus fontinalis Brook trout Salvelinus namaycush Lake trout GADIDAE Gadus morhua Atlantic cod ATHERINIDAE Odontesthes bonariensis Pejerrey SYNBRANCHIDAE Monopterus albus Lai CENTROPOMIDAE Centropomus undecimalis Common snook Lates calcarifer Barramundi/Asian sea bass Lates niloticus Nile perch PERCICHTHYIDAE Maccullochella peeli Murray cod Macquaria ambigua Golden perch Morone saxatilis Striped bass MORONIDAE Dicentrarchus labrax European seabass SERRANIDAE Epinephelus akaara Hong Kong grouper Epinephelus areolatus Areolate grouper Epinephelus tauvina Greasy grouper Plectropomus maculatus Spotted coralgrouper TERAPONTIDAE Bidyanus bidyanus Silver perch CENTRARCHIDAE Micropterus salmoides Largemouth black bass PERCIDAE Perca fluviatilis European perch Stizostedion lucioperca Pike-perch Perca fluvescens Yellow Perch Stizostedion canadense Sauger Stizostedion vitreum Walleye POMATOMIDAE Pomatomus saltatrix Bluefish CARANGIDAE Seriola dumerili Greater amberjack Seriola quinqueradiata Japanese amberjack Trachinotus blochii Snubnose pompano Trachinotus carolinus Florida pompano Trachinotus goodei Palometa pompano Trachurus japonicus Japanese jack mackerel RACHYCENTRIDAE Rachycentron canadum Cobia LUTJANIDAE Lutjanus argentimaculatus Mangrove red snapper Ocyurus chrysurus Yellowtail snapper SPARIDAE Acanthopagrus schlegeli Dark seabream Diplodus sargus White seabream Evynnis japonica Crimson seabream Pagrus major Red seabream Pagrus pagrus Red porgy Rhabdosargus sarba Goldlined seabream Sparus aurata Gilthead seabream SCIAENIDAE Sciaenops ocellatus Red drum CICHLIDAE Aequidens rivulatus Green terror Cichlasoma maculicauda Blackbelt cichlid Cichlasoma managuense Jaguar guapote Cichlasoma urophthalmus Mexican mojarra Etroplus suratensis Pearlspot Oreochromis andersonii Three spotted tilapia Oreochromis aureus Blue tilapia Oreochromis macrochir Longfin tilapia Oreochromis mossambicus Mozambique tilapia Oreochromis niloticus Nile tilapia Oreochromis spilurus Tilapia Oreochromis urolepis Wami tilapia Sarotherodon melanotheron Blackchin tilapia Tilapia guineensis Tilapia Tilapia rendalli Redbreast tilapia Tilapia zillii Redbelly tilapia MUGILIDAE Liza aurata Golden grey mullet Liza macrolepis Largescale mullet Liza parsia Gold-spot mullet Liza ramada Thinlip grey mullet Liza saliens Leaping mullet Liza tade Tade mullet Mugil cephalus Flathead grey mullet Mugil curema White mullet Mugil liza Lebranche mullet ELEOTRIDAE Dormitator latifrons Pacific fat sleeper Oxyeleotris marmorata Marble goby SIGANIDAE Siganus canaliculatus White-spotted spinefoot Siganus guttatus Goldlined spinefoot Siganus rivulatus Marbled spinefoot SCOMBRIDAE Thunnus maccoyii Southern bluefin tuna Thunnus thynnus Northern bluefin tuna ANABANTIDAE Anabas testudineus Climbing perch BELONTIIDAE Trichogaster pectoralis Snakeskin gourami HELOSTOMATIDAE Helostoma temmincki Kissing gourami OSPHRONEMIDAE Osphronemus goramy Giant gourami CHANNIDAE Channa argus Snakehead Channa micropeltes Indonesian snakehead Channa punctatus Spotted snakehead Channa striata Striped snakehead SCOPHTHALMIDAE Psetta maxima Turbot PARALICHTHYIDAE Paralichthys olivaceus Bastard halibut (Japanese flounder) Paralichthys dentatus Summer Flounder Paralichthys lethostigma Southern flounder Paralichthys americanus Winter flounder Hippoglossus hippoglossus Atlantic Halibut Rhombosolea tapirina Greenback flounder SOLEIDAE Solea vulgaris Common sole * And all hybrids between any of the above species.
[0065] As used herein, the term salmon refers to any species of the Family Salmonidae. Preferably, the salmon is a Salmo sp. or Oncorhynchus sp. More preferably, the salmon is a Salmo sp. Even more preferably, the salmon is Atlantic Salmon (Salmo salar).
[0066] In an embodiment, the fish, preferably salmon, is at a "larval" or "juvenile" stage. Fish development recognises 5 periods that occur in the following order: embryonic period; larval period; juvenile period; adult period; senescent period. The larval period occurs once the embryo has hatched and has the ability to feed independently of the egg yolk (or mother in rare cases), organ systems develop morphologically and gain physiological function. The juvenile period is when all organ systems are fully formed and functional (bar the gonads) and fish attain the appearance of miniature adults, the period lasts until the gonads become mature. Once the gonads mature the fish attain the adult period, and then senescence when growth ceases and gonads do not produce gametes (Adapted from Moyle, P. B. & Cech, J. J. 2004. Fishes An Introduction to Ichthyology, 5th Edition, Prentice Hall).
[0067] The "crustacean" may be any organism of the subphylum "Crustacea", and hence the crustacean may be obtained from marine sources and/or freshwater sources. Such crustacea include, but are not limited to, organisms such as krill, clams, shrimp (including prawns), crab, and lobster. Further examples of crustacea that can be reared on feedstuffs of the invention are provided in Table 2. The invention may be practised with any, all, or any combination of the listed crustacea.
TABLE-US-00002 TABLE 2 Crustacea that can be fed feedstuffs of the invention. Family Scientific name Common name PENAEIDAE Metapenaeus dobsoni Kadal shrimp Metapenaeus endeavouri Endeavour shrimp Metapenaeus ensis Greasyback shrimp Metapenaeus monoceros Speckled shrimp Penaeus aztecus Northern brown shrimp Penaeus chinensis Fleshy prawn Penaeus esculentus Brown tiger prawn Penaeus indicus Indian white prawn Penaeus japonicus Kuruma prawn Penaeus kerathurus Caramote prawn Penaeus merguiensis Banana prawn Penaeus monodon Giant tiger prawn Penaeus notialis Southern pink shrimp enaeus paulensis Sao Paulo shrimp Penaeus penicillatus Redtail prawn Penaeus schmitti Southern white shrimp Penaeus semisulcatus Green tiger prawn Penaeus setiferus Northern white shrimp Penaeus stylirostris Blue shrimp Penaeus subtilis Southern brown shrimp Penaeus vannamei Whiteleg shrimp Xiphopenaeus kroyeri Atlantic seabob SERGESTIDAE Acetes japonicus Akiami paste shrimp PALAEMONIDAE Macrobrachium Monsoon river prawn malcolmsonii Macrobrachium rosenbergii Giant river prawn Palaemon serratus Common prawn NEPHROPIDAE Homarus americanus American lobster Homarus gammarus European lobster ASTACIDAE Astacus astacus Noble crayfish Astacus leptodactylus Danube crayfish Jasus edwardsii Southern rock lobster Jasus lalandii Western rock lobster Pacifastacus leniusculus Signal crayfish CAMBARIDAE Procambarus clarkii Red swamp crawfish PARASTACIDAE Cherax destructor Yabby crayfish Cherax quadricarinatus Red claw crayfish Cherax tenuimanus Marron crayfish PALINURIDAE Panulirus longipes Longlegged spiny lobster PORTUNIDAE Portunus trituberculatus Gazami crab Scylla serrata Indo-Pacific swamp crab POTAMIDAE Eriocheir sinensis Chinese river crab * And all hybrids between any of the above species. indicates data missing or illegible when filed
Feedstuffs
[0068] For purposes of the present invention, "feedstuffs" include any food or preparation, for fish or crustacean consumption.
[0069] The present invention provides a feedstuff comprising lipid, the fatty acid of said lipid comprising at least 5.5% (w/w) stearidonic acid (SDA). The invention also provides methods of using said feedstuff for rearing a fish or crustacean.
[0070] In embodiments of the invention, the fatty acid of said lipid comprises at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11.0%, at least 15%, at least 20%, or at least 30% (w/w) SDA.
[0071] In further embodiments, the fatty acid of said lipid comprises less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or more preferably less than 8% (w/w) total saturated fatty acids (SFA). In particular, the feedstuff comprises reduced levels of 14:0 and/or 16:0 compared to the corresponding feedstuff made with fishoil rather than plant oil comprising at least 5.5% SDA.
[0072] Although the level of SDA that may be produced in seedoil of transgenic plants may be in excess of 40% of the fatty acid, the invention may be practised with plant oil that has less SDA, such as for example at least 5.5% SDA. That is, not all of the ALA is converted to SDA in the plant, and the oil may contain both SDA and ALA. Therefore, in yet other embodiments, the fatty acid of said lipid comprises at least 10%, at least 15%, at least 16%, at least 17%, at least 18%, or at least 19% (w/w) α-linolenic acid (ALA 18:3Δ9,12,15, ω3). In an embodiment, the ALA level is in the range 10-45% (w/w).
[0073] Preferably, the lipid of the feedstuff comprises phytosterol, which may provide additional benefit. In embodiments of the invention, the lipid comprises at least 0.025%, at least 0.05%, or at least 0.1% (w/w) phytosterol. It may comprise at least 0.2% phytosterol, typically in the range 0.2-0.8% (w/w) phytosterol. The phytosterol may be any plant derived sterol from plants such as, but not limited to, Echium sp., canola, soybean, flax, cereal or grain legume. Examples of phytosterols include, but are not limited to, brassicasterol, campesterol, stigmasterol, β-sitosterol or any combination of these.
[0074] In a further embodiment, the lipid is substantially free of cholesterol, which may be advantageous in limiting the cholesterol level in the fish or crustacean that is produced, in particular for fish. As used herein, the term "substantially free of cholesterol" refers to the lipid comprising less than 0.1% (w/w) cholesterol, preferably at an undetectable level. Typically, lipid obtained from plants is substantially free of cholesterol.
[0075] In other embodiments, at least 25%, at least 50%, at least 75% or at least 90% of the SDA is esterified in the form of triacylglycerol.
[0076] In yet further embodiments, the lipid content of the feedstuff is at least 10, at least 15, at least 20, at least 30, at least 50, at least 100, at least 200, or at least 250 g/kg dry matter. In another embodiment, the lipid content of the feedstuff is no more than 350 g/kg dry matter or any range between these figures.
[0077] In other embodiments, the feedstuff comprises at least 0.55, at least 1, at least 2.5, at least 5, at least 7.2, at least 10, at least 12.5, or more preferably at least 14.3 g/kg dry matter of SDA.
[0078] In yet another preferred embodiment, the fatty acid of the lipid content of the feedstuff comprises less than 2% EPA and/or DHA, more preferably less than 1% EPA and/or DHA.
[0079] The SDA can be from any source. In a preferred embodiment, the SDA is provided in the form of a transgenic organism, or extract or portion thereof, wherein the organism is genetically modified such that it produces SDA and/or produces higher levels of SDA than when compared to a wild-type organism. Preferably, the transgenic organism is a plant or yeast. In a particularly preferred embodiment, the SDA is provided in the form of oil extracted from a plant, especially a transgenic plant. Typically, such oil is extracted from the seed of the plant. However, in some embodiments, the SDA may be obtained from a non-transgenic organism which naturally produces SDA, for example, Echium plantagineum.
[0080] Fish and crustaceans can be fed feedstuffs of the present invention in any manner and amount, and according to any feeding schedule employed in fish or crustacean cultivation. Feeding rates typically vary according to abiotic factors, mainly seasonal such as temperature, and biotic, in particular the size of the animal. Juvenile fish are typically fed 5-10% of their body weight per day over about 4-6 feeds per day. Larger fish are typically fed at 2-5% of their body weight per day over about 1-2 feeds per day. Juvenile crustaceans may fed up to 5-10% of their body weight over about 6 feeds per day, while larger crustaceans may be fed a minimum of about 2% of their body weight per day over about 2-3 feeds per day. The fish or crustaceans may be allowed to feed to appetite.
[0081] Preferably, the fish or crustaceans are fed at least once per day, more preferably two or more times per day such as, for example, 2-6 or 4-6 times per day. It is preferred that any excess food be removed after the feeding period, e.g., by flushing out of a race-way system, or through removal out of the bottom of the sea-cage. Alternatively, a fish such as catfish can be added to the fish population to consume any excess food.
[0082] The benefits increase when fish or crustacean are fed over longer periods of time, for example over at least 6, 7 or 12 weeks. However, it would be appreciated that there is some benefit when the fish or crustacean is provided with the feedstuff over shorter time periods, relative to feeding the fish or crustacean feedstuff containing plant oil not comprising substantial SDA. Feedstuffs other than those described herein may also be used in the time period, however it is preferred that the feedstuff of the invention is used predominantly over the time period if not exclusively.
[0083] As used herein, "predominantly" means at least 50% of the time, occasions or in amount, as the context determines.
[0084] It is preferable that fish or crustaceans be fed SDA containing feedstuffs as a mixture with other well-known ingredients included in commercial fish or crustaceans food formulations so as to provide a nutritionally balanced complete food, including, but not limited to, plant matter, e.g., flour, meal, starch or cracked or processed grain produced from a crop plant such as wheat or other cereals, alfalfa, corn, oats, potato, rice, soybeans or other legumes; cellulose in a form that may be obtained from wood pulp, grasses, plant leaves, and waste plant matter such as rice or soy bean hulls, or corn cobs; animal matter, e.g., fish or crustacean meal, oil, protein or solubles and extracts, krill, meat meal, bone meal, feather meal, blood meal, or cracklings; algal matter; yeast; bacteria; vitamins, minerals, and amino acids; organic binders or adhesives; and chelating agents and preservatives. A wide variety of formulations are reported in both the patent and scientific literature. Alternatively, SDA is used to supplement other foods, e.g., commercial fish or crustacean foods.
[0085] In one embodiment, the feedstuff comprises fishmeal (which may or may not be defatted) but does not comprise, as a separate ingredient, fish oil. Alternatively, the feedstuff may comprise some fishoil as an added separate ingredient. However, the minimum level of SDA in the fatty acid of the total lipid of the feedstuff should remain at least 5.5%.
[0086] On a commercial scale feedstuffs may conveniently be provided in the form of pressed or extruded feed pellets.
[0087] The components utilized in the feedstuff compositions of the present invention can be of semi-purified or purified origin. By semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.
[0088] With respect to vitamins and minerals, the following may be added to the feedstuff compositions of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Examples of these include Stay C which is a commercial stabilised vitamin C product, trisodium phosphate or Banox E which is an antioxidant. Other such vitamins and minerals may also be added.
Desaturases
[0089] Organisms useful for producing feedstuffs of the invention typically comprise a gene encoding a Δ6 desaturase, which may be a transgene or an endogenous gene. As used herein, a "Δ6 desaturase" is at least capable of converting ALA to SDA, and/or linoleic acid (LA, 18:2Δ9,12, ω6) to γ-linolenic acid (GLA, 18:2Δ9,12, ω6). Examples of suitable Δ6 desaturases include, but are not limited to, those which comprises (i) an amino acid sequence as provided as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, (ii) an amino acid sequence which is at least 50% identical to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, or (iii) a biologically active fragment of i) or ii). In a further embodiment, the Δ6 desaturase comprises an amino acid sequence which is at least 90% identical to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. In a further embodiment, the Δ6 desaturase is encoded by the protein coding region of one of the Δ6 desaturase genes listed in Table 3 or gene at least 75% identical thereto.
[0090] The Δ6 desaturase may also have other activities such as Δ5 desaturase activity. Such enzymes are known in the art as a "Δ5/Δ6 bifunctional desaturase" or a "Δ5/Δ6 desaturase". These enzymes are at least capable of i) converting ALA to SDA, and ii) converting eicosatetraenoic acid to eicosapentaenoic acid. A gene encoding a bifunctional Δ5-/Δ6-desaturase has been identified from zebrafish (Hasting et al., 2001). The gene encoding this enzyme might represent an ancestral form of the "front-end desaturase" which later duplicated and the copies evolved distinct Δ5- and Δ6-desaturase functions. In one embodiment, the Δ5/Δ6 bifunctional desaturase is naturally produced by a freshwater species of fish. In a particular embodiment, the Δ5/Δ6 bifunctional desaturase comprises
[0091] i) an amino acid sequence as provided in SEQ ID NO:13,
[0092] ii) an amino acid sequence which is at least 50% identical to SEQ ID NO:13, or
[0093] iii) a biologically active fragment of i) or ii).
TABLE-US-00003 TABLE 3 Examples of Δ6 desaturases from different sources. Protein Type of Accession size organism Species Nos. (aa's) References Mammals Homo sapiens NM_013402 444 Cho et al., 1999; Leonard et al., 2000 Mus musculus NM_019699 444 Cho et al., 1999 Nematode Caenorhabditis elegans Z70271 443 Napier et al., 1998 Plants Borago officinales U79010 448 Sayanova et al., 1997 Echium AY055117 Garcia-Maroto et al., 2002 AY055118 Primula vialii AY234127 453 Sayanova et al., 2003 Anemone leveillei AF536525 446 Whitney et al., 2003 Mosses Ceratodon purpureus AJ250735 520 Sperling et al., 2000 Marchantia polymorpha AY583463 481 Kajikawa et al., 2004 Physcomitrella patens Girke et al., 1998 Fungi Mortierella alpina AF110510 457 Huang et al., 1999; AB020032 Sakuradani et al., 1999 Pythium irregulare AF419296 459 Hong et al., 2002 Mucor circinelloides AB052086 467 Rhizopus sp. AY320288 458 Zhang et al., 2004 Saprolegnia diclina 453 WO02081668 Diatom Phaeodactylum tricornutum AY082393 477 Domergue et al., 2002 Bacteria Synechocystis L11421 359 Reddy et al., 1993 Algae Thraustochytrium aureum 456 WO02081668 Fish Danio rerio AF309556 444 Hastings et al., 2001
[0094] Organisms useful in producing feedstuffs of the invention generally comprise a gene encoding an "ω3 desaturase", which may be a transgene or an endogenous gene. As used herein, an "ω3 desaturase" is at least capable of converting LA to ALA and/or GLA to SDA and are therefore able to introduce a desaturation at the third carbon-carbon bond from the co end of the acyl substrate. Such desaturases may also be known in the art as Δ15 desaturases when active on a C18 substrate, for example 18:2 (LA), introducing a desaturation at the fifteenth carbon-carbon bond from the carboxy (Δ) end of the acyl chain. Examples of ω3 desaturase include those described by Pereira et al. (2004), Horiguchi et al. (1998), Berberich et al. (1998) and Spychalla et al. (1997) or as listed in Table 4. Examples of suitable Δ15 desaturases include, but are not limited to, those which comprise (i) an amino acid sequence as provided in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29, (ii) an amino acid sequence which is at least 50% identical to any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29, or (iii) a biologically active fragment of i) or ii). In a further embodiment, the Δ15 desaturase comprises an amino acid sequence which is at least 90% identical to any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29. In a further embodiment, the Δ15 desaturase has an amino acid sequence according to an Accession No listed in Table 4, or is encoded by the protein coding region of one of the Δ15 desaturase genes listed in Table 4, or a protein or gene at least 75% identical thereto.
TABLE-US-00004 TABLE 4 Examples of ω3/Δ15 desaturases. Type of Accession Protein organism Species Nos. size References Plant Arabidopsis thaliana NP_850139.1 288 NCBI AY096462. 386 NCBI AAL77744 435 NCBI Brassica napus P48642 383 Arondel et al., 1992 AY599884 383 NCBI JQ2337 377 NCBI AAT65204 378 NCBI Brassica rapa subsp. oleifera AAL08867 302 Tanhuanpaa et al., 2002 Glycine max BAB18135 380 NCBI AAO24263 376 Bilyeu et al., 2003 P48621 453 Yadav et al., 1993 Linum usitatissimum ABA02173 391 Vrinten et al., 2005 ABA02172 392 Vrinten et al., 2005 Betula pendula AAN17504 386 NCBI Perilla frutescens AAD15744 391 Chung et al., 1999 AAL36934 390 NCBI AAB39387 438 NCBI Pelargonium × hortorum AAC16443 407 NCBI Malus × domestica AAS59833 439 NCBI Vernicia fordii CAB45155 387 NCBI AAD13527 437 Tang et al., 1999 Vigna radiata P32291 380 Yamamoto et al., 1992 Prunus persica AAM77643 449 NCBI Brassica juncea CAB85467 429 NCBI Nicotiana tabacum P48626 379 Hamada et al., 1994 BAA11475 441 Hamada et al., 1996 Betula pendula AAN17503 444 NCBI Zea mays BAA22442 398 Berberich et al., 1998 BAA22441 443 Berberich et al., 1998 Petroselinum crispum AAB72241 438 Kirsch et al., 1997 Sesamum indicum P48620 447 NCBI Helianthus annuus AAP78965 443 NCBI Capsicum annuum AAF27933 438 NCBI Ricinus communis P48619 460 VandeLoo et al., 1994 Sorghum bicolor AAT72937 389 Yang et al., 2004 Oryza sativa XP_479619 387 NCBI Solanum tuberosum CAA07638 431 NCBI Solanum lycopersicum AAP82169 435 Li et al., 2003 Triticum aestivum BAA28358 383 Horiguchi et al., 1998 Algae Chlorella vulgaris BAB78717 418 Suga et al., 2002 Synechococcus sp AAB61352 350 Sakamoto et al., 1997 Dunaliella salina AAD48897 196 NCBI Fungi Saprolegnia diclina AAR20444 358 Pereira et al., 2004 NCBI indicates sequences are available from http://www.ncbi.nlm.nih.gov/
[0095] The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence and a sequence defined herein are aligned over their entire length.
[0096] The term "polypeptide" is used interchangeably herein with the terms "protein" and "enzyme".
[0097] With regard to the defined polypeptides/enzymes, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the polypeptide comprises an amino acid sequence which is at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.
[0098] As used herein, the term "biologically active fragment" refers to a portion of the defined polypeptide/enzyme which still maintains desaturase activity. Such biologically active fragments can readily be determined by serial deletions of the full length protein, and testing the activity of the resulting fragment.
Cells
[0099] Suitable cells for use in feedstuffs of the invention, or which can be used to produce SDA for feedstuffs of the invention, include any cell containing SDA or that can be transformed with a polynucleotide encoding a polypeptide/enzyme described herein, and which is thereby capable of being used for producing SDA. Host cells into which the polynucleotide(s) are introduced can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Such nucleic acid molecule may be related to SDA synthesis, or unrelated. Host cells either can be endogenously (i.e., naturally) capable of producing proteins described herein or can be capable of producing such proteins only after being transformed with at least one nucleic acid molecule.
[0100] The cells may be prokaryotic or eukaryotic. Host cells can be any cell capable of producing SDA, and include fungal (including yeast), parasite, arthropod, animal and plant cells. Preferred host cells are yeast and plant cells. In a preferred embodiment, the plant cells are seed cells.
[0101] In one embodiment, the cell is an animal cell or an algal cell. The animal cell may be of any type of animal such as, for example, a non-human animal cell, a non-human vertebrate cell, a non-human mammalian cell, or cells of aquatic animals such as fish or crustacea, invertebrates, insects, etc.
[0102] The cells may be of an organism suitable for fermentation. Suitable fermenting cells, typically microorganisms are able to ferment, i.e., convert, sugars, such as glucose or maltose, directly or indirectly into the desired fermentation product. Examples of fermenting microorganisms include fungal organisms, such as yeast. As used herein, "yeast" includes Saccharomyces spp., Saccharomyces cerevisiae, Saccharomyces carlbergensis, Candida spp., Kluveromyces spp., Pichia spp., Hansenula spp., Trichoderma spp., Lipomyces starkey, and Yarrowia lipolytica.
Gene Constructs and Vectors
[0103] Transgenic organisms, and/or host cells, producing SDA are typically transformed with a recombinant vector. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
[0104] One type of recombinant vector comprises a nucleic acid molecule which encodes an enzyme useful for the purposes of the invention (such as a polynucleotide encoding a Δ6 desaturase or ω3 desaturase) operatively linked to an expression vector. As indicated above, the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of a desired nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells, including in bacterial, fungal, endoparasite, arthropod, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in yeast, animal or plant cells.
[0105] In particular, expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of desired nucleic acid molecules. In particular, recombinant molecules include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells. A variety of such transcription control sequences are known to those skilled in the art.
[0106] Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Transgenic Plants and Parts Thereof
[0107] The term "plant" as used herein as a noun refers to whole plants, but as used as an adjective refers to any substance which is present in, obtained from, derived from, or related to a plant, such as for example, plant organs (e.g. leaves, stems, roots, flowers), single cells (e.g. pollen), seeds, plant cells and the like. Plants provided by or contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. In preferred embodiments, plant useful for the production of feedstuffs of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, or pea), or other legumes. The plants may be grown for production of edible roots, tubers, leaves, stems, flowers or fruit. The plants of the invention may be: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolour, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), cassava (Manihot esculenta), coconut (Cocos nucifera), olive (Olea europaea), oats, or barley.
[0108] In one embodiment, the plant is an oilseed plant, preferably an oilseed crop plant. As used herein, an "oilseed plant" is a plant species used for the commercial production of oils from the seeds of the plant. The oilseed plant may be oil-seed rape (such as canola), maize, sunflower, soybean, sorghum, oil palm or flax (linseed). Furthermore, the oilseed plant may be other Brassicas, cotton, peanut, poppy, mustard, castor bean, sesame, safflower, or nut producing plants. The plant may produce high levels of oil in its fruit, such as olive or coconut.
[0109] Examples of cotton of the, and/or useful for, the present invention include any species of Gossypium, including, but not limited to, Gossypium arboreum, Gossypium herbaceum, Gossypium barbadense and Gossypium hirsutum.
[0110] When the production of SDA is desired it is preferable that the plant species which is to be transformed has an endogenous ratio of ALA to LA which is at least 1:1, more preferably at least 2:1. Examples include most, if not all, oilseeds such as linseed. This maximizes the amount of ALA substrate available for the production of SDA. This may be achieved by transgenic means; for example by introduction of a Δ15 desaturase gene into the plant to increase the levels of the ALA substrate for conversion into SDA.
[0111] The plants produced for use in feedstuffs of the invention may already be transgenic, and/or transformed with additional genes to those described in detail herein.
[0112] Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Leguminous plants include beans, peas, soybeans, lupins and the like. Beans include guar, locust bean, fenugreek, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
[0113] The term "extract or portion thereof" refers to any part of the plant. "Portion" generally refers to a specific tissue or organ such as a seed or root, whereas an "extract" typically involves the disruption of cell walls and possibly the partial purification of the resulting material. Naturally, the "extract or portion thereof" will comprise SDA. Extracts can be prepared using standard techniques of the art.
[0114] Transgenic plants, as defined in the context of the present invention include plants and their progeny which have been genetically modified using recombinant techniques. This would generally be to cause or enhance production of at least one protein/enzyme defined herein in the desired plant or plant organ. Transgenic plant parts include all parts and cells of said plants such as, for example, cultured tissues, callus, protoplasts. Transformed plants contain genetic material that they did not contain prior to the transformation. The genetic material is preferably stably integrated into the genome of the plant. Such plants are included herein in "transgenic plants". A "non-transgenic plant" is one which has not been genetically modified with the introduction of genetic material by recombinant DNA techniques. In a preferred embodiment, the transgenic plants are homozygous for each and every gene that has been introduced (transgene) so that their progeny do not segregate for the desired phenotype.
[0115] Several techniques exist for introducing foreign genetic material into a plant cell. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (see, for example, U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may be transformed using Agrobacterium technology (see, for example, U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135). Electroporation technology has also been used to transform plants (see, for example, WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO 92/09696 and WO 93/21335). In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during development and/or differentiation using appropriate techniques known to those skilled in the art.
[0116] A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
[0117] Examples of plant promoters include, but are not limited to ribulose-1,6-bisphosphate carboxylase small subunit, beta-conglycinin promoter, phaseolin promoter, high molecular weight glutenin (HMW-GS) promoters, starch biosynthetic gene promoters, ADH promoter, heat-shock promoters and tissue specific promoters. Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency. Typical enhancers include but are not limited to Adh-intron 1 and Adh-intron 6.
[0118] Constitutive promoters direct continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these promoters may also be used.
[0119] In a particularly preferred embodiment, the promoter directs expression in tissues and organs in which lipid and oil biosynthesis take place, particularly in seed cells such as endosperm cells and cells of the developing embryo. Promoters which are suitable are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baumlein et al., 1991), 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), the linin gene promoter from flax, or the legumin B4 promoter (Baumlein et al., 1992), and promoters which lead to the seed-specific expression in monocots such as maize, barley, wheat, rye, rice and the like. Notable promoters which are suitable are the barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) or the promoters described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene, the rye secalin gene). Other promoters include those described by Broun et al. (1998) and US 20030159173.
[0120] Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress. Other desirable transcription and translation elements that function in plants may be used.
[0121] In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S) and the like may be used.
EXAMPLES
Example 1
Materials and Methods
Lipid Extraction and Isolation
[0122] Samples were freeze dried and extracted using a modified Bligh and Dyer protocol (Bligh and Dyer, 1959). A single phase extraction, CHCl3/MeOH/H2O, (1:1:0.9, by vol), was used to yield a total lipid extract (TLE).
[0123] Lipid classes were analysed by an Iatroscan MK V thin-layer chromatography-flame ionization detector (TLC-FID) analyser (Iatron Laboratories, Japan). Samples were spotted onto silica gel SIII Chromarods (5 μm particles size) and developed in a glass tank lined with pre-extracted filter paper. The solvent system used for the lipid separation was hexane: diethyl ether: acetic acid (60:17:0.1, v/v/v) (Volkman and Nichols, 1991). After development for 25 minutes, the chromarods were oven-dried and analysed immediately to minimise adsorption of atmospheric contaminants. Lipid classes were quantified by DAPA software (Kalamunda, WA, Australia). The FID was calibrated for each compound class: phosphatidylcholine; cholesterol; cholesteryl ester; oleic acid; hydrocarbon (squalene); wax ester (derived from fish oil); triacylglycerol (derived from fish oil); and DAGE (purified from shark liver oil).
[0124] An aliquot of the TLE was trans-methylated in methanol:chloroform:hydrochloric acid (10:1:1, v/v/v) for 1 hour at 100° C. After addition of water the mixture was extracted three times with hexane: chloroform (4:1, v/v) to produce fatty acid methyl esters (FAME). FAME were concentrated under nitrogen and treated with N,O-bis(trimethylsilyl)-trifloroacetamide (BSFTA, 50 μl, 60° C., 1 h) to convert hydroxyl groups to their corresponding trimethylsilyl ethers. Samples were made up to a known volume with an internal injection standard (23:0 or 19:0 FAME) and analysed by gas chromatography (GC) using an Agilent Technologies 6890N GC (Palo Alto, Calif., USA) equipped with an HP-5 cross-linked methyl silicone fused silica capillary column (50 m×0.32 mm i.d.), and an FID. Helium was used as the carrier gas. Samples were injected, by a split/splitless injector and an Agilent Technologies 7683 Series auto sampler in splitless mode, at an oven temperature of 50° C. After 1 min the oven temperature was raised to 150° C. at 30° C. min-1, then to 250° C. at 2° C. per min and finally to 300° C. at 5° C. min-1. Peaks were quantified by Agilent Technologies GC ChemStation software (Palo Alto, Calif., USA). Individual components were identified by mass spectral data and by comparing retention time data with those obtained for authentic and laboratory standards. GC results are typically subject to an error of ±5% of individual component area. GC-mass spectrometric (GC-MS) analyses were performed on a Finnigan Thermoquest GCQ GC-mass spectrometer fitted with an on-column injector with Thermoquest Xcalibur software (Austin, Tex., USA). The GC was fitted with a capillary column similar to that described above.
[0125] A polar column was used to separate 18:1ω9 and 18:3ω3 which coeluted on the HP5 column. FAME were analysed with a Hewlett Packard 5890 gas chromatograph (GC) equipped with a flame ionisation detector (FID) at 250° C. FAME samples were injected using a split/splitless injector into a polar BPX-70 fused-silica column (50 m×0.32 mm i.d.). The carrier gas was helium. The GC oven temperature was initially held at 45° C. for 2 min after injection and then increased at 30° C./min to 120° C. and at 3° C./min to 240° C., then held isothermal for 10 min.
Statistical Analysis
[0126] Mean values were reported plus or minus standard error of the mean. Percentage data were arcsin transformed prior to analysis. Normality and homogeneity of variance were confirmed and a comparison between means was achieved by 1-way analysis of variance (ANOVA). Multiple comparisons were achieved by Turkey-Kramer HSD. Significance was accepted as probabilities of 0.05 or less. Statistical analysis was performed using SPSS for windows version 11.
Brassica Transformation
[0127] Brassica napus (Line BLN 1239) seeds were surface sterilized by soaking them in 70% (v/v) ethanol for 2 min and then rinsed for 10 min in tap water at 55° C. The seeds were sterilized for 20 min in 25% commercial bleach (10 gl-1 sodium hypochlorite) containing 0.1% Tween-20. The seeds were washed thoroughly with sterile distilled H2O, placed on GEM medium in tissue culture jars and kept in the cold room for two days for germination. The jars were transferred to low light (20 μMm2s-1) for about four to six days at 24° C. for growth of the cotyledons. Roots and apices were removed under asceptic conditions. Excised hypocotyl segments (10 mm) were washed with 50 ml CIM medium for about 30 min without agitation in the laminar flow cabinet. The CIM was removed and the segments transferred to a 250 ml flask with 50 ml of CIM, sealed with sterile aluminium foil and shaken for 48 hours at 24° C. under low light (10 μMm2s-1).
[0128] Agrobacterium strains containing plasmid transformation vectors were grown in 5 ml of LB media with appropriate antibiotics at 28° C. for about two days, transferred to a 250 ml Erlenmeyer flask with 45 ml of LB without antibiotics and cultured for four hours at 28° C. with shaking. The Agrobacterium cells were pelleted by centrifugation, washed, and gently re-suspended in about 20 ml BM. The optical density at 600 nm of the resultant Agrobacterium suspension was adjusted to 0.2 with BM. The cell suspension was added to the explants which had been drained of the CIM medium, mixed briefly and allowed to stand for 20 min. The Agrobacterium suspension was removed, the hypocotyl explants washed once with 50 ml CIM and co-cultivation continued for 48 hours on an orbital shaker. After this, the medium was slightly milky due to Agrobacterium growth. CIM was removed and the explants washed three times with 50 ml CIM for one minute and then twice for one hour on an orbital shaker at 140×g. Following the washes, 50 ml CIM containing 200 mg/l Timentin® was added and placed on an orbital shaker for 24 hours. Under sterile conditions, the CIM medium was clear at this stage.
[0129] Regeneration of transformed shoots on SIM was carried out on a two-stage selection process. Initially, the hygromycin concentration in the SIM medium used was 5 mg/l. After about two weeks, explants with developing calli were transferred to SIM containing 20 mg/l hygromycin. When the regenerating shoots had developed leaves longer than one cm, they were excised carefully and were transferred to SEM with 20 mg/l hygromycin. After two weeks, stems usually had elongated and apices were transferred to RIM containing 10 mg/l hygromycin. Non-elongating shoots were sub-cultured in SEM every two to three weeks until they were long enough to be transferred to RIM. When the roots were about two cm in length, the regenerated plantlets were removed from tissue culture pots and transferred to soil for further growth.
Media Recipes
[0130] Composition of the tissue culture media used in this procedure is given below. They contained MS salts (Murashige and Skoog, 1962), MS or B5 vitamins (Gamborg et al., 1968), sucrose and MES. The pH was adjusted to 5.8 with KOH prior to sterilization. For solid media, agar was added and then autoclaved. Media containing agar was allowed to cool to below 50° C. and filter-sterilized compounds were added to the melted media before pouring it into either plastic Petri dishes or 250 ml polycarbonate tissue culture jars (Sarstedt, No 75.9922519). The composition of various media with all additives are given below: germination medium (GEM); basal medium (BM); callus-inducing medium (CIM, modified from Radke et al., 1988); washing medium (WM); shoot-inducing medium (SIM, modified from Radke et al., 1988); shoot-elongation medium (SEM) and root-inducing medium (RIM, modified from De Block et al., 1989).
[0131] GEM: 1×MS salts, 1×MS vitamins, Sucrose (20 gl-1), MES (500 mgl-1), Agar (8 gl-1), pH to 5.8.
[0132] BM: 1×MS salts, 1×B5 vitamins, Sucrose (30 gl-1), MES (500 mgl-1), pH to 5.8.
[0133] CIM: 2,4-D (1.0 mgl-1) and Kinetin (1.0 mgl-1) added to BM.
[0134] WM: 2,4-D (1.0 mgl-1), Kinetin (1.0 mgl-1) and Timentin® (200 mgl-1) added to BM.
[0135] SIM: AgNO3 (500 mgl-1), Zeatin riboside (0.5 mgl-1), BAP (2.0 mgl-1), GA3 (0.01 mgl-1), Timentin® (200 mgl-1), Hygromycin (5 to 30 mgl-1), and Agar (8 gl-1) added to BM.
[0136] SEM: 0.5×MS salts, 0.5×B5 vitamins, Sucrose (10 gl-1), MES (500 mgl-1), Timentin® (200 mgl-1), Hygromycin (20 to 30 mgl-1), Agar (8 gl-1), pH to 5.8.
[0137] RIM: 0.5×MS salts, 0.5×B5 vitamins, Sucrose (10 gl-1), MES (500 mgl-1), IBA (0.1 mgl-1), Timentin® (200 mgl-1), Hygromycin (20 to 30 mgl-1), Agar (8 gl-1), pH to 5.8.
Example 2
Fish Fed with Food Compositions Including Plant-Derived SDA
[0138] Stearidonic acid (SDA, 18:4ω3) is an LC-PUFA precursor, derived by desaturation of ALA by Δ6 desaturase (FIG. 1). The Δ6 desaturase is also involved other steps in the biosynthesis of LC-PUFA in the formation of DHA from EPA in vertebrates (Yamazaki et al., 1992) and 18:2 ω6 to 20:4 ω6. Therefore it is possible that the Δ6 desaturation of ALA is out-competed by the Δ6 pathway in fish and crustacea when diets contain high levels of 18:2 ω6, present in vegetable oils such as canola and sunflower.
[0139] Oil from a few plant sources such as Echium plantagineum have SDA in the fatty acid profile, up to about 15-20% as a percentage of the fatty acid in the oil. To determine whether SDA-rich oil might serve as an efficient substrate for ω3 LC-PUFA accumulation in fish, a feeding trial was conducted in vivo using salmon (Salmo salar L.). Diets including an equivalent level of canola oil were used as a control source of ALA, as described in Tables 5 and 6.
TABLE-US-00005 TABLE 5 Ingredient and lipid composition (g/kg dry matter) of experimental diets. Diet CO SO Mix FO oil (g) oil (g) oil (g) oil (g) Ingredient composition (g kg-1) Fishmeal (defattened) 150 150 150 150 Casein 150 150 150 150 Wheat Gluten 100 100 100 100 Hipro soy 226 226 226 226 Fish oil 0 0 0 130 Canola oil 130 0 65 0 SDA oil 0 130 65 0 Pre Gel Starch 150 150 150 150 Vitamin Mixa 3 3 3 3 Mineral Mixb 5 5 5 5 Stay Cc 3 3 3 3 Choline chloride 2 2 2 2 Bentontie 50 50 50 50 CMC 10 10 10 10 Sodium Mono P 20 20 20 20 Yttrium Oxide 10 10 10 10 FAME Total SFA 6.7 10.8 12.2 44.9 Total MUFA 81.2 41.3 56.2 32.9 18:3ω3 ALA 13.1 25.4 20.9 3.1 18:4ω3 SDA 0.0 14.3 7.2 4.2 20:5ω3 EPA 0.1 0.1 0.0 18.0 22:6ω3 DHA 0.6 0.4 0.0 10.7 Total ω3 13.9 40.2 28.6 39.6 18:2ω6 28.2 25.8 27.0 8.0 Total ω6 28.2 26.1 27.0 9.3 Other PUFA 0.0 11.6 5.9 3.3 Total PUFA 42.1 77.9 61.5 52.2 SO, stearidonic rich oil crossential SA14 from Croda chemicals; CO, canola oil diet; Mix, 1:1 mix diet of canola oil and stearidonic acid oil; FO, fish oil diet, SFA, Saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; CMC, Carboxymethyl cellulose; DHA, Docosahexaenoic Acid; EPA, SDA, Stearidonic acid; Eicosapentaenoic Acid. aVitamin mix (ASV4) to supply per kilogram feed: 2.81 mg thiamin HCL, 1.0 mg riboflavin, 9.15 mg pyridoxine HCL, 25 mg nicotinic acid, 54.35 mg calcium D-pantothenate, 750 mg myo-inositol, 0.38 mg D-biotin, 2.5 mg folic acid, 0.03 mg cyanocobalamin, 6350 IU retinol acetate, 2800 IU cholecalciferol, 100 IU DL quadrature-tocopherol acetate, 5 mg menadone sodium bisulphate, 100 mg Roche rovimix E50. bMineral mix (TMV4) to supply per kilogram feed: 117 mg CuSO4•5H2O, 7.19 mg KI, 1815 mg FeSO4•7H2O, 307 mg MnSO4•H2O, 659 mg ZnSO4•7H2O, 3.29 mg Na2SeO3, 47.7 mg CoSO4•7H2O cL-Ascorbyl-2-polyphosphate (Stay-C, Roche Vitamins Australia, French Forest, NSW, Australia).
[0140] Four diets were formulated to compare canola oil (CO), two different levels of stearidonic acid oil (100% (SO), 1:1 SO:CO (Mix)), and fish oil (FO) (Tables 5 and 6). Fish meal was defattened three times using a 2:1 mixture of hexane and ethanol (400 ml 100 g-1 fish meal). Soybean (Hamlet Protein A/S, Horsens, Denmark), casein (MP Biomedcals Australasia Pty Ltd, Seven Hills NSW, Australia), wheat gluten (Starch Australasia, Land Cove, NSW, Australia) and BOIIC pre-gelatinised maize starch (Penford Australia Limited, Lane Cove, NSW, Australia) were used. Stearidonic acid rich oil was provided as Crossential SA14 (Croda Chemicals, East Yorkshire, UK). Fish oil was from jack mackerel (Skretting Australia, Cambridge, Tasmania Australia). Stay-C and Rovimix E50 were supplied from Roche Vitamins Australia (Frenchs Forest, NSW, Australia), and the remaining ingredients were supplied by Sigma-Aldrich (Castle Hill, NSW, Australia). Yttrium Oxide was used as a digestibility marker. The diets were manufactured into 3 mm diameter pellets using a California Pellet Mill (CL-2), dried and stored at -5° C.
[0141] The feeding experiment was conducted at the School of Aquaculture, University of Tasmania, Launceston, Australia. Atlantic salmon (Salmo salar) parr were obtained from Wayatinah Salmon hatchery (SALTAS, Tasmania, Australia) and randomly stocked into 300 l tanks at 25 fish per tank. They were acclimated for 10 days. The tanks were held at a constant temperature of 15.0° C. and a photoperiod of 16:8 (light:dark). The fish were held in a partial freshwater recirculation system. Water was treated through physical, UV and biofilters, with a continuous replacement of approximately 15% per day. Dissolved oxygen, pH, ammonia, nitrate, nitrite, and chlorine were monitored daily to ensure water quality remained within parameters recommended for Atlantic salmon (Wedemeyer, 1996).
TABLE-US-00006 TABLE 6 Fatty acid composition of the lipid in the diets (% of total fatty acid). FA CO SE SO SE Mix SE FO SE 14:0 0.23 0.00 0.13 0.02 0.21 0.01 6.38 0.08 16:0 1.58 0.79 4.30 1.24 5.57 0.93 19.23 0.20 18:0 2.58 0.01 3.83 0.02 3.19 0.01 3.90 0.04 Other Sat 0.75 0.01 0.06 0.00 0.44 0.00 5.02 0.01 Total Sat 5.13 8.33 9.40 34.53 16:1ω7 0.28 0.00 0.17 0.03 0.25 0.00 7.06 0.05 18:1ω9 52.03 0.17 24.45 0.06 37.54 0.06 10.88 0.19 18:1ω7 3.28 0.02 1.04 0.02 2.18 0.02 2.69 0.01 20:1ω9 0.96 0.00 0.74 0.01 0.87 0.00 1.66 0.01 Other Mono 5.92 0.06 5.32 0.17 2.42 0.12 3.02 0.03 Total Mono 62.47 31.73 43.26 25.31 18:3ω3 10.07 0.03 19.57 0.04 16.04 0.03 2.39 0.04 18:4ω3 0.00 0.00 11.01 0.09 5.57 0.03 3.20 0.06 20:4ω3 0.00 0.00 0.00 0.00 0.00 0.00 0.73 0.01 20:5ω3 0.05 0.02 0.05 0.02 0.00 0.00 13.85 0.12 22:5ω3 0.14 0.04 0.00 0.00 0.00 0.00 1.46 0.02 22:6ω3 0.43 0.01 0.33 0.06 0.41 0.01 8.26 0.08 Other ω3 0.00 0.00 0.00 0.00 0.00 0.00 0.59 0.00 Total ω3 10.68 30.96 22.01 30.46 18:2ω6 21.71 0.04 19.82 0.03 20.81 0.01 6.18 0.10 18:3ω6 0.00 0.00 8.20 0.06 4.33 0.02 0.64 0.06 20:3ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 20:4ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.80 0.01 22:5ω6 0.00 0.00 0.00 0.00 0.00 0.00 0.19 0.03 Other ω6 0.00 0.00 0.23 0.04 0.00 0.00 0.00 0.00 Total ω6 21.71 28.25 25.13 7.82 Other PUFA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Total PUFA 32.40 59.21 47.15 38.28
[0142] Fish were initially anaesthetized (50 mg benzocaine) and weights and lengths were recorded. Four fish were killed and assessed for initial lipid content and composition. Twenty five fish were randomly allotted into twelve 300 l tanks. Fish weights were not significantly different between tanks (43.6 g±0.7). The four diets were fed in triplicate on a ration of 1.1% body weight per day (% BW d-1), in two equal feeds at 0900 and 1700 hrs by automatic belt feeders. Every three weeks all fish were anaesthetized (50 mg l-1, benzocaine) and weighed. Fish were starved the day prior to measuring. Every 7 days the total feed consumption (kg DM) was estimated from the amount of feed that was not eaten by collection in sediment collectors. The amount of uneaten feed was estimated from the number of uneaten pellets using the average weight of a pellet from each feed (Helland et al., 1996).
[0143] Specific growth rates (SGR) were calculated as
SGR (% day-1)=100×(ln(W2/w1))×d-1
where W1 and W2 were the weights (g) at the two times and d was the number of days.
[0144] At the end of the experiment fish were starved for one day prior to being anaesthetized (50 mg l-1, benzocaine) and their weight and fork length measured. Three fish per tank were killed by a blow to the head after immersion in anaesthetic. Samples of tissue were dissected with red muscle and white muscle sampled below the dorsal fin. Samples were frozen at -80° C. until analysis.
Results
[0145] No significant difference was found between fish fed the four diets with respect to initial and final weight, weight gain, specific growth rate (SPR), total feed consumption (FC), feed efficiency ratio (FER), hepatosomatic index (HSI) or survival as determined using ANOVA (Table 7).
[0146] After 42 days there was no statistical difference in the composition of flesh lipid with respect to the lipid classes for the different dietary groups, in either red or white muscle (Tables 8 and 9). The predominant lipid class in red muscle was TAG (94.0-96.7%). There was significantly (p>0.02) less TAG in the fed fish (42.0-67.0%) compared to the initial measurement (82.0%) for the white muscle.
[0147] For fatty acid composition, there were significantly (p>0.01) higher levels of 18:3ω3 and 18:4ω3, in both white and red muscle tissues, in the fish fed SO than in fish fed the Mix diet. Both 18:3ω3 and 18:4ω3 levels were significantly higher than in the FO and CO fed fish (Tables 8 and 9). There were significantly (p>0.01) higher levels in both muscle tissues of 22:6ω3 and total ω3 in the FO and SO diets compared to the Mix and CO diets. There were significantly (p>0.01) higher levels of 20:5ω3 in the FO and SO fed fish compared to the CO fed fish in both the red and white muscle. The ratio of ω3/ω6 was significantly (p>0.01) lower in the CO and Mix diet fed fish compared to the SO and FO diets.
TABLE-US-00007 TABLE 7 Growth and efficiencies of Atlantic salmon fed experimental feeds with Canola oil (CO), Stearidonic acid rich oil (SO), 1:1 CO:SO (Mix) and Fish oil (FO) (mean ± SE). Feed CO Mix SO FO Initial weight (g) 46.2 ± 2.5 44.6 ± 1.1 44.8 ± 1.1 42.3 ± 1.2 Final Weight (g) 81.4 ± 8.4 80.1 ± 1.9 76.9 ± 2.2 76.5 ± 3.3 Weight gain (g) 35.1 ± 5.9 35.5 ± 0.8 32.1 ± 2.0 34.1 ± 3.1 SGR (% day-1) 1.2 ± 0.2 1.3 ± 0.0 1.2 ± 0.1 1.2 ± 0.1 Total FC (g DM) 41.4 ± 2.0 41.9 ± 0.8 40.5 ± 0.7 38.0 ± 1.8 FER (g/g DM) 0.8 ± 0.1 0.8 ± 0.0 0.8 ± 0.1 0.9 ± 0.0 HSI (%) 1.0 ± 0.1 1.0 ± 0.1 0.9 ± 0.2 0.9 ± 0.1 Survival 98.7 ± 1.4 98.7 ± 1.4 100.0 ± 0.0 100.0 ± 0.0 SO, stearidonic rich oil diet; CO, canola oil diet; Mix, 1:1 mix diet of canola oil and stearidonic acid rich oils; FO, fish oil diet; DM, Dry matter 1 SGR, Specific growth rate = 100 × (ln (Wfinal(g)/Winitial(g))) × number of days (d)-1 2FC, Total feed consumption = Total amount (g DM) consumed by an individual over 42 days. 3FER, feed efficiency ratio = total weight gain (g)/total feed consumption (g DM). 4HSI, hepatosomatic index = 100 (liver weight (g WW)/Total body weight (g WW)). Survival during growth experiment.
[0148] In both muscle tissues, the FO diet surprisingly provided significantly (p>0.01) higher levels of 14:0, 16:0 and total saturates compared with CO and Mix fed. The FO diet also provided significantly (p>0.01) higher levels of 14:0 in both muscle tissues and 16:0 and total saturates in the red muscle compared with the SO fed fish. In both muscle tissues, FO and SO fed salmon had significantly (p>0.01) lower levels of 18:1 ω9 and total MUFA compared to the fish fed CO and Mix diets. There was significantly (p>0.01) higher levels of 18:2 ω6 and total ω6 in the fish fed CO and Mix diets compared with FO fed fish.
TABLE-US-00008 TABLE 8 FAME Content and lipid class composition of total lipid of Red muscle samples of Atlantic salmon fed Canola Oil (CO) 1:1 mix of Canola Oil:Stearidonic oil (Mix), Stearidonic oil (SO) diets and Fish oil (FO) FAME Initial SE CO SE Mix SE SO SE FO SE Sig f 14:0 4.0 ± 0.3b 3.3 ± 0.2a 3.0 ± 0.2a 3.9 ± 0.1a 5.2 ± 0.2b 0.01 21.9 16:0 16.7 ± 0.4b,c 12.9 ± 0.2a 12.7 ± 0.3a 14.4 ± 0.4a,b 16.7 ± 0.3c 0.01 14.8 18:0 4.7 ± 0.3 4.2 ± 0.0 4.6 ± 0.1 4.5 ± 0.0 4.3 ± 0.1 Other SFA e 2.0 ± 0.0c 1.7 ± 0.0b 1.4 ± 0.0a 1.8 ± 0.0b 1.9 ± 0.0b,c 0.01 11.1 Total SFA 27.3 ± 0.7b,c 22.2 ± 0.9a 21.7 ± 0.4a 24.6 ± 1.0a,b 28.2 ± 0.4c 0.01 13.9 16:1ω7c 5.9 ± 0.4c,d 5.0 ± 0.2b,c 4.3 ± 0.4a 5.8 ± 0.2b,c 7.4 ± 0.4d 0.01 16.3 18:1ω9c 13.4 ± 0.6a 30.5 ± 1.3b 27.9 ± 1.1b 16.1 ± 0.5a 14.9 ± 0.2a 0.01 26.5 18:1ω7c 3.3 ± 0.1c,d 3.3 ± 0.0b,c 2.9 ± 0.1a 3.0 ± 0.1a,b 3.5 ± 0.1d 0.01 9.3 20:1ω9c 1.4 ± 0.1b 2.0 ± 0.0b 1.3 ± 0.4a,b 1.7 ± 0.0b 0.4 ± 0.4a 0.01 5.0 Other MUFA f 2.5 ± 0.0a 2.3 ± 0.0a 2.5 ± 0.0a 2.5 ± 0.0a 4.2 ± 0.0b 0.01 10.2 Total MUFA 26.4 ± 0.5a 43.2 ± 2.2b 38.9 ± 1.1b 29.1 ± 0.6a 30.5 ± 0.5a 0.01 28.2 18:3ω3 ALA 0.7 ± 0.0a 2.0 ± 0.1b 3.9 ± 0.2c 5.7 ± 0.2d 2.0 ± 0.0b 0.01 65.8 18:4ω3 SDA 2.3 ± 0.2a 2.2 ± 0.1a 3.7 ± 0.b 4.3 ± 0.3c 2.6 ± 0.1a 0.01 92.2 20:4ω3 1.1 ± 0.0a 1.0 ± 0.0a,b 1.2 ± 0.0b 1.4 ± 0.0c 1.2 ± 0.0a,b 0.01 10.4 20:5ω3 EPA 8.6 ± 0.2b 4.8 ± 0.3a 4.4 ± 0.3a 6.2 ± 0.2b 7.6 ± 0.3b 0.01 25.2 22:5ω3 DPA 3.2 ± 0.1c 2.3 ± 0.1a,b 2.2 ± 0.2a 3.1 ± 0.1b,c 3.7 ± 0.1c 0.01 11.0 22:6ω3 DHA 19.2 ± 1.0c 9.6 ± 0.5a 9.0 ± 0.7a 12.5 ± 0.6b 14.4 ± 0.7b 0.01 13.6 Other ω3 g 1.0 ± 0.0 0.8 ± 0.0 0.7 ± 0.0 1.1 ± 0.0 1.3 ± 0.0 Total ω3 36.2 ± 0.6b 22.6 ± 1.9a 25.0 ± 1.1a 34.3 ± 1.1b 32.8 ± 0.6b 0.01 16.3 18:2ω6 LA 2.8 ± 0.1a 7.6 ± 0.4b 9.1 ± 0.7b 6.2 ± 0.6a,b 3.9 ± 0.7a 0.01 12.7 18:3ω6 0.2 ± 0.0a 0.5 ± 0.0b 0.5 ± 0.0b 1.5 ± 0.2c 0.8 ± 0.2a,b 0.01 8.1 20:3ω6 0.2 ± 0.0a 0.6 ± 0.0b,c 1.0 ± 0.1c 0.7 ± 0.1c 0.2 ± 0.1a,b 0.01 12.5 20:4ω6 1.3 ± 0.2a.b 0.5 ± 0.0a 0.5 ± 0.0a 0.6 ± 0.0a,b 0.6 ± 0.0b 0.01 5.3 22:5ω6 0.3 ± 0.0b 0.2 ± 0.0a,b 0.2 ± 0.0a 0.3 ± 0.0a,b 0.3 ± 0.0b 0.01 5.5 Other ω6 h 0.8 ± 0.0 1.0 ± 0.0 0.8 ± 0.0 0.8 ± 0.0 0.9 ± 0.0 Total ω6 5.3 ± 0.2a 9.9 ± 0.9c 11.6 ± 0.8c 8.5 ± 1.2b,c 5.8 ± 0.1a,b 0.01 12.8 Other PUFA i 4.8 ± 0.2 2.0 ± 0.0 2.8 ± 0.1 3.4 ± 0.1 2.7 ± 0.1 Total PUFA 46.3 ± 1.3b 34.6 ± 2.1a 39.4 ± 1.3a,b 46.3 ± 1.2b 41.3 ± 0.9b 0.01 15.1 Ratios ω3/ω6 6.8 ± 0.3b 2.3 ± 0.4a 2.2 ± 0.2a 4.0 ± 0.1b 5.6 ± 0.2b 0.01 54.5 Lipid Class TAG 96.7 ± 0.4 96.7 ± 0.3 95.4 ± 0.2 96.6 ± 0.4 94.0 ± 0.9 FFA 0.7 ± 0.1a 0.7 ± 0.1a 1.8 ± 0.1a,b 0.5 ± 0.1a 2.5 ± 0.6b 0.01 8.0 ST 0.8 ± 0.2 1.1 ± 0.0 1.0 ± 0.1 1.0 ± 0.0 0.9 ± 0.4 PL 1.8 ± 0.2 1.5 ± 0.2 1.7 ± 0.2 1.8 ± 0.3 2.6 ± 0.3 mg/g Wetj 17.8 ± 1.0 22.9 ± 0.7 22.2 ± 1.1 24.5 ± 1.8 28.1 ± 5.5 mg/g Dryj 44.3 ± 2.8 53.6 ± 0.9 57.0 ± 7.7 54.1 ± 2.9 56.6 ± 7.1 SFA, Saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; DHA, Docosahexaenoic Acid; DPA, Docosapentaenoic Acid; EPA, Eicosapentaenoic Acid; SDA, Stearidonic acid; LA, Linoleic acid; ALA, α- Linolenic acid; TAG; Triacylglycerol; FFA, free fatty acid; ST, sterol; PL, polar lipid; WW, wet weight; Sig, Significance; f, Mean sum of squares. a,b,c,dMean values across the row not sharing a common letter were significantly different as determined by Turkey-Kramer HSD; df = 4.15. e Other SFA includes 15:0, 17:0, 20:0, 22:0 and 24:0 f Other MUFA includes 16:1ω9, 16:1ω5, 18:1ω5, 20:1ω7, 22:1ω9, 22:1ω11 and 24:1ω9 g Other ω3 PUFA include 21:5ω3 and 24:6ω3 h Other ω6 PUFA include 20:2ω6, 20:3ω6, 22:4ω6 and 24:5ω6 i Other PUFA include 16:2ω4, 16:3ω4 and 18:2ω9 jDetermined by TLC-FID
TABLE-US-00009 TABLE 9 FAME Content and lipid class composition of the total lipid of white muscle samples of Atlantic salmon FAME Initial SE CO SE Mix SE SO SE FO SE Sig f 14:0 3.9 ± 0.4b 2.3 ± 0.1a 1.9 ± 0.2a 2.3 ± 0.1a 3.6 ± 0.2b 0.01 13.0 16:0 18.3 ± 0.4a,b 14.8 ± 0.3a 15.2 ± 0.3a 16.8 ± 0.6a,b 19.7 ± 0.3b 0.01 5.0 18:0 5.7 ± 0.3 4.8 ± 0.0 5.6 ± 0.1 5.6 ± 0.1 5.0 ± 0.1 Other SFAe 1.8 ± 0.0 1.0 ± 0.0 0.8 ± 0.0 1.0 ± 0.0 1.4 ± 0.0 Total SFA 29.7 ± 1.1a,b 22.9 ± 0.9a 23.5 ± 1.7a 25.7 ± 1.4a,b 29.6 ± 0.5b 0.01 5.1 16:1ω7c 5.6 ± 0.5b 3.1 ± 0.2a 2.7 ± 0.4a 3.1 ± 0.1a 5.1 ± 0.4b 0.01 14.1 18:1ω9c 14.2 ± 0.7b 27.2 ± 1.3c 22.2 ± 1.3b,c 11.3 ± 0.8a 11.0 ± 6.7a 0.01 5.9 18:1ω7c 3.2 ± 0.1c 2.9 ± 0.0b,c 2.5 ± 0.1a,b 2.2 ± 0.1a 3.1 ± 0.1c 0.01 18.0 20:1ω9c 1.1 ± 0.2 1.5 ± 0.0 0.9 ± 0.4 1.0 ± 0.0 0.5 ± 0.4 Other MUFAf 3.3 ± 0.1 2.1 ± 0.0 2.0 ± 0.0 1.7 ± 0.0 2.4 ± 0.0 Total MUFA 27.3 ± 0.7b 36.8 ± 2.3c 30.2 ± 2.1b 19.3 ± 2.4a 22.0 ± 1.1a 0.01 4.7 18:3ω3 ALA 1.0 ± 0.0a 2.1 ± 0.1b 3.5 ± 0.7c 6.3 ± 0.4d 1.7 ± 0.1b 0.01 30.1 18:4ω3 SDA 2.0 ± 0.2a 1.6 ± 0.0a 2.8 ± 0.3a 3.9 ± 0.1b 2.0 ± 0.1a 0.01 10.8 20:4ω3 1.1 ± 0.0a,b 0.8 ± 0.0a 1.2 ± 0.0a,b 1.3 ± 0.1b 1.0 ± 0.0a,b 0.01 4.7 20:5ω3 EPA 7.4 ± 0.2b,c 4.8 ± 0.3a 5.4 ± 0.3a,b 7.3 ± 0.5b,c 8.6 ± 0.3c 0.01 7.0 22:5ω3 DPA 3.0 ± 0.1b,c 2.1 ± 0.1a 2.2 ± 0.2a 2.6 ± 0.1a,b 3.6 ± 0.2c 0.01 10.3 22:6ω3 DHA 20.0 ± 1.2a,b 16.2 ± 0.9a 18.3 ± 0.7a 22.2 ± 0.6b 24.2 ± 0.7b 0.01 8.0 Other ω3g 0.8 ± 0.1 0.5 ± 0.0 0.3 ± 0.0 0.6 ± 0.0 0.9 ± 0.0 Total ω3 35.4 ± 0.2b 28.2 ± 2.1a 33.7 ± 1.1a,b 44.2 ± 2.6c 42.0 ± 2.4b,c 0.01 14.4 18:2ω6LA 2.9 ± 0.2a 7.6 ± 0.4b 7.5 ± 0.7b 5.6 ± 0.6b 3.2 ± 0.7a 0.02 4.0 18:3ω6 0.6 ± 0.2a 0.5 ± 0.0a 0.9 ± 0.4a,b 1.5 ± 0.2b 0.4 ± 0.3a 0.01 12.0 20:3ω6 0.1 ± 0.0a 1.0 ± 0.1b 1.1 ± 0.1b 0.9 ± 0.2b 0.1 ± 0.1a 0.02 4.5 20:4ω6 1.3 ± 0.2 1.0 ± 0.0 1.3 ± 0.0 1.0 ± 0.1 0.9 ± 0.0 22:5ω6 0.2 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 Other ω6h 1.3 ± 0.2 0.9 ± 0.0 0.6 ± 0.0 0.5 ± 0.0 0.4 ± 0.0 Total ω6 5.9 ± 0.1a 10.8 ± 1.3b 10.6 ± 1.8b 8.3 ± 1.4a,b 4.7 ± 0.2a 0.02 6.2 Other PUFAi 1.7 ± 0.0 1.4 ± 0.0 1.9 ± 0.1 2.5 ± 0.1 1.7 ± 0.1 Total PUFA 43.0 ± 1.2a 40.4 ± 1.4a 46.3 ± 2.3a,b 55.0 ± 1.1c 48.4 ± 0.9b Ratios ω3/ω6 6.0 ± 0.1b,c 2.6 ± 0.5a 3.2 ± 0.1a 5.3 ± 0.1b 8.8 ± 0.2c 0.02 16.2 Lipid Class TAG 82.0 ± 2.6c 46.2 ± 4.9a 67.0 ± 4.6a 59.9 ± 2.0a 50.5 ± 12.7a 0.02 3.1 FFA 1.7 ± 0.3b,c 1.9 ± 0.2c 0.4 ± 0.1a 1.8 ± 0.1b,c 0.5 ± 0.2a,b 0.02 5.9 ST 2.1 ± 0.4 4.0 ± 0.2 2.1 ± 0.2 3.8 ± 0.3 2.2 ± 0.3 PL 14.2 ± 2.1 47.6 ± 4.9 30.5 ± 4.7 34.6 ± 1.9 46.8 ± 12.4 mg/g Wetj 8.4 ± 0.3 9.1 ± 0.1 9.0 ± 0.2 9.2 ± 0.1 8.2 ± 0.3 mg/g Dryj 10.1 ± 1.0a 15.2 ± 0.4b 14.2 ± 0.6b 14.9 ± 0.2b 15.1 ± 1.0b 0.02 8.9 Abbreviations and other footnote definitions, see Table 8.
DISCUSSION
[0149] The inclusion of SO at 130 or 65 g/kg of diet for Atlantic salmon parr did not significantly influence growth or feed conversion rates compared to other experimental diets during the 42 day growth trial in freshwater (Table 7). There was little effect between diets in the lipid class profiles (Tables 8 and 9). There was significantly less TAG in the white muscle of the fed fish compared to the diet due to the inclusion of oil in the diet at a level of 130 g/kg compared to the commercial diet (approx. 300 g/kg) they were fed pre-experiment.
[0150] Fish muscle FA profiles were closely related to the FA profile of their diet. It has been shown previously for salmon fed using canola, sunflower and linseed oils, i.e. diets rich in ALA and without EPA and DHA, that there was a significant reduction in total ω3 and ω3 LC-PUFA, in particular DHA and EPA (Bransden et al., 2003; Bell et al., 2003; Polvi and Ackman, 1992; Bell et al., 2004). Therefore, minimal conversion to, or negligible accumulation of, LC-PUFA occurred when fish were fed vegetable oil. In those studies growth rates and the health of fish fed vegetable oils were not affected.
[0151] In the study described here, Atlantic salmon parr sizes were initially 43.6 g±0.7 g to a final weight of 72.4 g±1.9 g. The fish were at an important stage of the growth. Pre-smoltification Atlantic salmon store FA, in particular ω3 LC-PUFA, prior to the energy requiring transfer to salt water, during which salmon undergo major changes in their lipid metabolism.
[0152] The inclusion of SDA at 14.3 or 7.2 g/kg significantly influenced the FA profiles of the salmon (Tables 8 and 9). Fish fed on the diet containing the higher level of SDA had significantly higher levels of EPA, DPA, DHA and total ω3 in the muscle samples than fish fed on the CO diet. In some respects, the fatty acid composition of the fish tissues was improved over that of fish fed the FO diet. For example, the level of saturated fat was reduced. The SO diet was also advantageous for this feature in combination with the high levels of LC-PUFA.
[0153] Neither the CO diet nor the SO diet contained EPA or DHA at substantial levels, being <0.7% of the fatty acid present in the lipid, the trace level probably originating with the fishmeal component. Therefore the increased accumulation of EPA; DPA and DHA in the fish tissues must have represented increased biosynthesis of the fatty acids from SDA in the fish.
[0154] This experiment showed that high levels of total ω3, DHA and EPA could be maintained in fish such as salmon without their inclusion as dietary FA. This experiment also demonstrated that the levels of fatty acids achieved, as reported in Tables 8 and 9, for example the levels of SDA, EPA, DPA, DHA, total LC-PUFA ω3, or total ω3 PUFA (includes C18 fatty acids), were minimum levels that could be achieved through feeding the fish a diet including plant derived SDA, and that even higher levels could be expected by using diets with even higher levels of SDA and/or longer feeding times.
[0155] The conversion of ALA to SDA involves the desaturation at the Δ6 position of the carbon chain with further chain elongation steps, followed by Δ5 desaturation to form EPA. The synthesis of EPA to DHA requires additional chain elongations and also involves the Δ6 desaturation in the conversion of 24:5 ω3 to 24:6 ω3 before chain shortening to DHA (FIG. 1); this is termed the Sprecher pathway. With the conversion of 18:2 ω6 to 20:4 ω6 also using the Δ6 desaturase, it was possible that the high levels of 18:2 ω6 in vegetable oils might compete for this enzyme and therefore minimal conversion of ALA to SDA would occur in the ω3 pathway. We have found here that this problem can be alleviated by adding SDA in the fish diet. The results indicated that a SDA rich plant oil could be used as a source of dietary oil for aquafeeds and, importantly, that the use of SDA oil did not affect the amount of ω3 LC-PUFA in the FA profile of salmon muscle.
Example 3
Prawn and Lobster Feedstuffs
[0156] For feeding of lobsters, prawns or other crustacean with diets high in SDA oil, the following feed compositions can be used (Table 10). Values provided as g/kg dry matter.
Example 4
Isolation of a Gene Encoding a Δ6-Desaturase from Echium plantagineuin
[0157] Some plant species such as evening primrose (Oenothera biennis), common borage (Borago officinalis), blackcurrant (Ribes nigrum), and some Echium species belonging to the Boragenacae family contain the ω6- and ω3-desaturated C18 fatty acids, γ-linolenic acid (18:3ω6, GLA) and stearidonic acid (18:4ω3, SDA) in their leaf lipids and seed TAG (Guil-Guerrero et al., 2000). GLA and SDA are recognized as beneficial fatty acids in human nutrition. The first step in the synthesis of LC-PUFA is a Δ6-desaturation. GLA is synthesized by a Δ6-desaturase that introduces a double bond into the Δ6-position of LA. The same enzyme is also able to introduce a double bond into Δ6-position of ALA, producing SDA. Δ6-desaturase genes have been cloned from members of the Boraginacae, like borage (Sayanova et al., 1997) and two Echium species (Garcia-Maroto et al., 2002).
TABLE-US-00010 TABLE 10 Prawn and Lobster feedstuffs. Spiny Lobster Prawn Fish meal (defatted) 250 0 Fish meal (standard) 0 200 Krill meal 0 185 Soybean Meal 150 150 Wheat gluten 100 100 Echium plantagineum Oil 110 100 Cholesterol 2 2 Lecithin 12 12 Pre-gel starch 175 100 Manucol 60 60 Vit Pre-Mix 2.00 2.00 Banox E 0.20 0.20 Choline Chloride 0.20 0.20 Vitamin C a 1.00 1.00 Carophyll pink 1.50 1.50 Min Pre-Mix b 0.01 0.01 TSP Phosphate 20.00 20.00 Mussel meal 50.00 0.00 Filler 66.00 66.00 Total 1000 1000 SDA 1.54 1.40 SO, stearidonic rich oil crossential SA14 from Croda chemicals; a L-Ascorbyl-2-polyphosphate (Stay-C, Roche Vitamins Australia, French Forest, NSW, Australia). b Mineral mix (TMV4) to supply per kilogram feed: 117 mg CuSO4•5H2O, 7.19 mg KI, 1815 mg FeSO4•7H2O, 307 mg MnSO4•H2O, 659 mg ZnSO4•7H2O, 3.29 mg Na2SeO3, 47.7 mg CoSO4•7H2O Soybean (Hamlet Protein A/S, Horsens, Denmark), wheat gluten (Starch Australasia, Land Cove, NSW, Australia) and BOIIC pre-gelatinised maize starch (Penford Australia Limited, Lane Cove, NSW, Australia) were used. Stay-C and Carophyll pink were supplied from Roche Vitamins Australia (Frenchs Forest, NSW, Australia), Mussel meal obtained from New Zealand Greenshell ® mussel, (Sealord P/L Nelson, New Zealand) and the remaining ingredients were supplied by Sigma-Aldrich (Castle Hill, NSW, Australia).
[0158] Echium plantagineum is a winter annual native to Mediterranean Europe and North Africa. Its seed oil is unusual in that it has a unique ratio of ω3 and ω6 fatty acids and contains high amounts of GLA (9.2%) and SDA (12.9%) (Guil-Guerrero et al., 2000), suggesting the presence of Δ6-desaturase activity involved in desaturation of both ω3 and ω6 fatty acids in seeds of this plant.
Cloning of E. plantagineum EplD6Des Gene
[0159] Degenerate primers with built-in XbaI or SacI restriction sites corresponding to N- and C-termini amino acid sequences MANAIKKY (SEQ ID NO:14) and EALNTHG (SEQ ID NO:15) of known Echium pitardii and Echium gentianoides (Garcia-Maroto et al., 2002) L6-desaturases were used for RT-PCR amplification of Δ6-desaturase sequences from E. platangineum using a proofreading DNA polymerase Pfu Turbo® (Stratagene). The 1.35 kb PCR amplification product was inserted into pBluescript SK(+) at the XbaI and SacI sites to generate plasmid pXZP 106. The nucleotide sequence of the insert was determined. It comprised an open reading frame encoding a polypeptide of 438 amino acid residues (SEQ ID NO:10) which had a high degree of homology with other reported Δ6-desaturases from E. gentianoides(SEQ ID NO:11), E. pitardii (SEQ ID NO:12) and Borago officinalis (SEQ ID NO:4). It has a cytochrome b5 domain at the N-terminus, including the HPGG (SEQ ID NO:16) motif in the heme-binding region, as reported for other Δ6- and Δ8-desaturases (Sayanova et al. 1997; Napier et al. 1999). In addition, the E. plantagineum Δ6 desaturase contains three conserved histidine boxes present in majority of the `front-end` desaturases (Napier et al., 1999). Cluster analysis including representative members of Δ6 and Δ8 desaturases showed a clear grouping of the cloned gene with other Δ6 desaturases especially those from Echium species.
[0160] Heterologous Expression of E. plantagineum Δ5-Desaturase Gene in Yeast
[0161] Expression experiments in yeast were carried out to confirm that the cloned E. platangineum gene (cDNA sequence provided as SEQ ID NO:25) encoded a Δ6-desaturase enzyme. The gene fragment was inserted as an XbaI-SacI fragment into the SmaI-SacI sites of the yeast expression vector pSOS (Stratagene) containing the constitutive ADH1 promoter, resulting in plasmid pXZP271. This was transformed into yeast strain S288Cα by a heat shock method and transformant colonies selected by plating on minimal media plates. For the analysis of enzyme activity, 2 mL yeast clonal cultures were grown to an O.D.600 of 1.0 in yeast minimal medium in the presence of 0.1% NP-40 at 30° C. with shaking. Precursor free-fatty acids, either linoleic or linolenic acid as 25 mM stocks in ethanol, were added so that the final concentration of fatty acid was 0.5 mM. The cultures were transferred to 20° C. and grown for 2-3 days with shaking. Yeast cells were harvested by repeated centrifugation and washing first with 0.1% NP-40, then 0.05% NP-40 and finally with water. Fatty acids were extracted and analyzed. The peak identities of fatty acids were confirmed by GC-MS.
[0162] The transgenic yeast cells expressing the Echium EplD6Des were able to convert LA and ALA to GLA and SDA, respectively. Around 2.9% of LA was converted to GLA and 2.3% of ALA was converted to SDA, confirming the Δ6-desaturase activity encoded by the cloned gene.
[0163] Functional Expression of E. platangineum Δ6-Desaturase Gene in Transgenic Tobacco
[0164] In order to demonstrate that the EplD6Des gene could confer the synthesis of Δ6 desaturated fatty acids in transgenic plants, the gene was expressed in tobacco plants. To do this, the gene fragment was excised from pXZP106 as an XbaI-SacI fragment and cloned into the plant expression vector pBI121 (Clonetech) at the XbaI and SacI sites under the control of a constitutive 35S CaMV promoter, to generate plant expression plasmid pXZP341. This was introduced into Agrobacterium tumefaciens AGL1, and used for transformation of tobacco W38 plant tissue, by selection with kanamycin.
[0165] Northern blot hybridization analysis of transformed plants was carried out to detect expression of the introduced gene, and total fatty acids present in leaf lipids of wild-type tobacco W38 and transformed tobacco plants were analysed as described above. Untransformed plants contained appreciable amounts of LA (21% of total fatty acids) and ALA (37% of total fatty acids) in leaf lipids. As expected, neither GLA nor SDA, products of Δ6-desaturation, were detected in the untransformed leaf. Furthermore, transgenic tobacco plants transformed with the pBI121 vector had similar leaf fatty acid composition to the untransformed W38 plants. In contrast, leaves of transgenic tobacco plants expressing the EplD6Des gene showed the presence of additional peaks with retention times corresponding to GLA and SDA. The identity of the GLA and SDA peaks were confirmed by GC-MS. Notably, leaf fatty acids of plants expressing the EplD6Des gene consistently contained approximately a two-fold higher concentration of GLA than SDA even when the total Δ6-desaturated fatty acids amounted up to 30% of total fatty acids in their leaf lipids (Table 11).
TABLE-US-00011 TABLE 11 Fatty acid composition in lipid from transgenic tobacco leaves (%). Total Δ6-de- saturated Plant 16:0 18:0 18:1 18:2 GLA 18:3 SDA products W38 21.78 5.50 2.44 21.21 -- 37.62 -- -- ET27-1 20.33 1.98 1.25 10.23 10.22 41.10 6.35 16.57 ET27-2 18.03 1.79 1.58 14.42 1.47 53.85 0.48 1.95 ET27-4 19.87 1.90 1.35 7.60 20.68 29.38 9.38 30.07 ET27-5 15.43 2.38 3.24 11.00 0.84 49.60 0.51 1.35 ET27-6 19.85 2.05 1.35 11.12 4.54 50.45 2.19 6.73 ET27-8 19.87 2.86 2.55 11.71 17.02 27.76 7.76 24.78 ET27-11 17.78 3.40 2.24 12.62 1.11 51.56 0.21 1.32 ET27-12 16.84 2.16 1.75 13.49 2.71 50.80 1.15 3.86
[0166] Northern analysis of multiple independent transgenic tobacco lines showed variable levels of the EplD6Des transcript which generally correlated with the levels of Δ6-desaturated products synthesized in the plants. For example, transgenic plant ET27-2 which contained low levels of the EplD6Des transcript synthesised only 1.95% of its total leaf lipids as Δ6-desaturated fatty acids. On the other hand, transgenic plant ET27-4 contained significantly higher levels of EplD6Des transcript and also had a much higher proportion (30%) of Δ6-desaturated fatty acids in its leaf lipids.
[0167] Analysis of the individual tobacco plants showed that, without exception, GLA was present at a higher concentration than SDA even though a higher concentration of ALA than LA was present in untransformed plants. In contrast, expression of EplD6Des in yeast had resulted in approximately equivalent levels of conversion of LA into GLA and ALA into SDA. Echium plantagineum seeds, on the other hand, contain higher levels of SDA than GLA. EplD6Des probably carries out its desaturation in vivo in Echium plantagineum seeds on LA and ALA esterified to phosphatidyl choline (PC) (Jones and Harwood 1980). In the tobacco leaf assay, the enzyme is most likely desaturating LA and ALA esterified to the chloroplast lipid monogalactosyldiacylglyerol (MGDG) (Browse and Slack, 1981). In the yeast assay, free fatty acid precursors LA and ALA added to the medium most likely enter the acyl-CoA pool and are available to be acted upon by EplD6Des in this form.
[0168] In conclusion, the transgenic tobacco plant described herein can be used to produce feedstuffs of the invention.
Functional Expression of E. platangineum Δ6-Desaturase Gene in Transgenic Seed
[0169] To show seed-specific expression of the Echium Δ6-desaturase gene, the coding region was inserted into the seed-specific expression cassette as follows. An NcoI-SacI fragment including the Δ6-desaturase coding region was inserted into pXZP6, a pBluescriptSK derivative containing a Nos terminator, resulting in plasmid pXZP157. The SmaI-ApaI fragment containing the coding region and terminator EplD6Des-NosT was cloned into pWVec8-Fpl downstream of the Fpl prompter, resulting in plasmid pXZP345. The plasmid pXZP345 was used for transforming wild type Arabidopsis plants, ecotype Columbia, and transgenic plants selected by hygromycin B selection. The transgenic plants transformed with this gene were designated "DP" plants.
[0170] Fatty acid composition analysis of the seed oil from T2 seed from eleven T1 plants transformed with the construct showed the presence of GLA and SDA in all of the lines, with levels of Δ6-desaturation products reaching to at least 11% (Table 12). This demonstrated the efficient Δ6-desaturation of LA and ALA in the seed.
Example 5
Transformation of Flax with a Seed-Specific Echium Δ6 Fatty Acid Desaturase Gene Construct
[0171] The full protein coding region of the Echium Δ6 fatty acid desaturase gene was PCR amplified with the following primers incorporating an XhoI site at the both ends: Ed6F: 5'-ACTCGAGCCACCATGGCTAATGCAATCAA-3' (SEQ ID NO:17) and Ed6R: 5'-CCTCGAGCTCAACCATGAGTATTAAGAG-3' (SEQ ID NO:18). PCR was conducted by heating to 94° C. for 2 min, followed by 30 cycles of 94° C. for 40 sec, 62° C. for 40 sec and 72° C. for 1 min 20 sec. After the last cycle, reactions were incubated for 10 min at 72° C. The PCR fragment was cloned into a pGEMTeasy® vector (Promega) and sequenced to ensure that no PCR-induced errors had been introduced. The insert was then digested with XhoI and inserted into the XhoI site of the binary vector, pWBVec8, in a sense orientation between the promoter derived from a seed-specifically expressed flax 2S storage protein gene, linin, and its polyadenylation site/transcription terminator.
TABLE-US-00012 TABLE 12 Fatty acid composition in transgenic Arabidopsis seeds expressing Δ6-desaturase from Echium. Fatty acid (%) Total Δ6- Plant 18:2.sup.Δ9, 12 18:3.sup.Δ6, 9, 12 18:3.sup.Δ9, 12, 15 18:4.sup.Δ6, 9, 12, 15 desaturation Columbia 16:0 18:0 18:1.sup.Δ9 (LA) (GLA) (ALA) (SDA) 20:0 20:1 products (%) DP-2 8.0 2.8 22.9 27.3 2.5 11.3 0.7 1.6 15.8 3.2 DP-3 7.8 2.7 20.6 25.9 3.0 12.1 0.8 1.7 17.8 3.8 DP-4 7.8 2.8 20.4 28.5 1.2 13.7 0.4 1.7 16.1 1.5 DP-5 8.2 3.2 17.4 29.3 1.2 14.2 0.3 2.1 15.6 1.6 DP-7 8.2 2.9 18.4 26.7 5.0 12.7 1.4 1.7 15.2 6.4 DP-11 9.0 3.5 17.8 28.4 3.0 13.4 0.9 2.1 13.9 3.8 DP-12 8.6 3.0 18.9 27.8 3.3 12.6 1.0 1.8 15.4 4.3 DP-13 8.7 2.9 14.4 27.3 8.5 13.7 2.6 1.7 12.4 11.1 DP-14 9.3 2.9 14.2 32.3 2.1 15.4 0.7 1.8 12.8 2.8 DP-15 8.2 2.9 17.8 30.1 0.3 15.3 0.2 1.9 15.5 0.5 DP-16 8.0 2.8 19.5 29.2 2.7 13.1 0.8 1.7 14.2 3.5
[0172] The binary vector, pWBVec8 contained a hygromycin resistance gene as a selectable marker for plant transformation (Wang et al., 1998). The construct, designated pVLin-Ed6 and containing the Echium Δ6 desaturase gene for seed-specific expression was shown schematically in FIG. 2. The linin promoter (SEQ ID NO:23) and terminator (SEQ ID NO:24) have previously been shown to confer expression in a highly specific manner in developing flax embryos, being expressed maximally in flax seed at the same time as oil accumulation in flax seeds. Both the linin promoter and terminator elements were able to drive seed specific expression of transgenes in flax at levels comparable to the highly active bean phaseolin promoter.
[0173] Approximately 150 hypocotyls were excised from 6-7 day old seedlings of flax cultivar Ward grown in sterile condition on MS media. This cultivar was found to produce the highest transformation efficiency among many flax cultivars, however many other cultivars were also amendable for gene transformation. The hypocotyls were inoculated and co-cultivated with Agrobacterium tumefaciens strain AGL1 harbouring the binary construct pVLin-Ed6 in a similar fashion to that described for Brassica transformation in Example 1. Following a co-cultivation period of 3-4 days at 24° C., the hypocotyls were transferred onto selection medium which was MS medium containing 200 mg/l Cefotaxime, 10 mg/l hygromycin, 1 mg/l BAP (6-benzyl-aminopurine) and 0.1 mg/l NAA (napthaleneacetic acid). Shoot development was initiated after about 2 weeks. Shoots were transferred onto fresh MS medium with the same additives except NAA was reduced to 0.02 mg/l. After 2-3 weeks, healthy green shoots were transferred onto fresh MS media without growth regulators for induction of roots. Rooted shoots were planted in potting mix in glasshouse.
[0174] The transgenic nature of regenerated flax plants was confirmed by PCR amplification of part of the Echium Δ6 fatty acid desaturase sequence with the primers Ed6s1, 5'-ACTCTGTTTCTGAGGTGTCCA-3' (SEQ ID NO:19); and Ed6a1, 5'-CATATTAACCCTAGCCATACACAT-3' (SEQ ID NO:20). DNA extracted from individual, regenerated flax plants was used as template in PCR reactions using the following amplification conditions: denaturation at 94° C. for 2 min, followed by 30 cycles of 94° C. for 40 sec, 58° C. for 40 sec and 72° C. for 1 min. Seeds set on forty primary transgenic flax plants will be analysed for the presence of SDA and GLA using lipid extraction followed by gas chromatography. It is expected that high levels of SDA will be produced in many of the plants and that SDA levels will be greater than GLA levels.
[0175] Seed from the transformed flax plants or extracts such as the oil or the seed meal can be used in feed compositions for use in feeding fish or crustacea.
Example 6
Transformation of Cotton with a Seed-Specific Construct Expressing an Echium Δ6 Fatty Acid Desaturase Gene
[0176] Cottonseed normally contains only negligible amounts (<0.5% of total fatty acids) of α-linolenic acid (ALA). In order to produce ALA at increased levels in cottonseed oil, cotton (Gossypium hirsutum) was transformed with a seed-specific gene construct expressing a FAD3 gene from Brassica napus (Arondel et al., 1992) (encoded protein amino acid sequence provided as SEQ ID NO:27). The accession number of the cDNA clone of this gene was L01418. The full protein coding region of the B. napus FAD3 gene was amplified by PCR using the primers BnFAD3-S1, 5'-CTCCAGCGATGGTTGTTGCTAT-3' (SEQ ID NO:21) and BnFAD3-A1, 5'-AATGTCTCTGGTGACGTAGC-3' (SEQ ID NO:22). The PCR product was cloned into a pGEMTeasy® vector (Promega) and the excised by restriction digest with NotI. The B. napus FAD3 coding sequence was inserted in the sense orientation into the NotI site between the soybean lectin gene promoter and terminator sequences (Cho et al., 1995), to provide a seed-specific expression construct. This vector contained an NPTII gene conferring kanamycin resistance as a selectable marker for plant transformation. This vector was introduced into Agrobacterium and used to transform cotton as described in Liu et al (2002). Independent transgenic plants expressing the FAD3 gene were obtained and lines accumulating ALA retained.
[0177] Separate cotton transformation experiments were performed using a similar seed-specific lectin cassette expressing a Δ6 fatty acid desaturase, to convert LA to GLA and ALA to SDA. The full protein-coding region of the Δ6 desaturase from Echium plantagineum (Zhou et al., 2006; SEQ ID NO:25) was amplified by PCR using the following primers incorporating a SmaI site at the 5' end, and SacI at the 3' end. Ed6F: 5'-ATCCCCGGGTACCGGTCGCCACCATGGCTAATGCAATCAAGAAGTA-3' (SEQ ID NO:30) and Ed6R: 5'-TTGGAGCTCAACCATGAGTATTAAGAGCTTC-3' (SEQ ID NO:31). The PCR fragment was cloned into pGEM-Teasy® vector (Promega) and sequenced to ensure no PCR-induced errors were introduced. The PCR amplified Δ6 desaturase gene was subsequently cloned into the corresponding SmaI/SacI sites in a sense orientation behind the napin (Fpl) promoter and upstream of the nos3' terminator-polyadenylation signal. Agrobacterium tumefaciens strain AGL1 harbouring the resulted construct, pGNapin-E6D, was used to transform cotton variety Coker315 by the method described by Liu et al. (2002).
[0178] Nine fertile independently transformed plants were obtained. The transformed cotton plants were positive for the presence of the transgene, and expression in developing seeds, by PCR and Northern blot analysis of the expressed RNA. 15 individual mature seeds from each of these primary transgenic plants were subjected to the analysis of fatty acid composition using gas chromatography (GC) as described above. Surprisingly high levels of γ-linolenic acid (GLA) were found to accumulate in four transgenic lines, while there was no detectable GLA in the non-transformed control plants. Levels of GLA of greater than 15% were observed in many seeds, and the level reached greater than 25% in some seeds that were likely to be homozygous for the introduced Δ6 desaturase gene. The accumulation of GLA is mainly at the expense of linoleic acid. Indeed, the conversion of LA to GLA (measured as % GLA×100/(% LA+% GLA) in the seedoil) was highly efficient in these cottonseeds relative to seeds of other plants, being greater than 25% in many seed and reaching in excess of 45% in some seed.
[0179] Cotton lines containing both genes will be produced by crossing the transformants expressing the FAD3 gene and transformants expressing the Δ6 desaturase gene, to produce lines containing SDA. By the methods described above, oilseed plants such as cotton or flax may be produced which produce at least 5.5% SDA on a weight basis in the fatty acid of the seed oil. Preferably, the level of SDA in the fatty acid is at least 11%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% on a weight basis. The efficiency of conversion of ALA to SDA (measured as % SDA×100/(% ALA+% SDA) in the seedoil) is at least 25% and preferably at least 45%. That is, at least 25%, preferably at least 45% of the polyunsaturated fatty acid in the cotton or flax seed that has a carbon chain of C18 or longer is desaturated at the Δ6 position.
[0180] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
[0181] All publications discussed above are incorporated herein in their entirety.
[0182] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.
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Sequence CWU
1
1
311444PRTHomo sapiens 1Met Gly Lys Gly Gly Asn Gln Gly Glu Gly Ala Ala Glu
Arg Glu Val 1 5 10 15
Ser Val Pro Thr Phe Ser Trp Glu Glu Ile Gln Lys His Asn Leu Arg
20 25 30 Thr Asp Arg Trp
Leu Val Ile Asp Arg Lys Val Tyr Asn Ile Thr Lys 35
40 45 Trp Ser Ile Gln His Pro Gly Gly Gln
Arg Val Ile Gly His Tyr Ala 50 55
60 Gly Glu Asp Ala Thr Asp Ala Phe Arg Ala Phe His Pro
Asp Leu Glu 65 70 75
80 Phe Val Gly Lys Phe Leu Lys Pro Leu Leu Ile Gly Glu Leu Ala Pro
85 90 95 Glu Glu Pro Ser
Gln Asp His Gly Lys Asn Ser Lys Ile Thr Glu Asp 100
105 110 Phe Arg Ala Leu Arg Lys Thr Ala Glu
Asp Met Asn Leu Phe Lys Thr 115 120
125 Asn His Val Phe Phe Leu Leu Leu Leu Ala His Ile Ile Ala
Leu Glu 130 135 140
Ser Ile Ala Trp Phe Thr Val Phe Tyr Phe Gly Asn Gly Trp Ile Pro 145
150 155 160 Thr Leu Ile Thr Ala
Phe Val Leu Ala Thr Ser Gln Ala Gln Ala Gly 165
170 175 Trp Leu Gln His Asp Tyr Gly His Leu Ser
Val Tyr Arg Lys Pro Lys 180 185
190 Trp Asn His Leu Val His Lys Phe Val Ile Gly His Leu Lys Gly
Ala 195 200 205 Ser
Ala Asn Trp Trp Asn His Arg His Phe Gln His His Ala Lys Pro 210
215 220 Asn Ile Phe His Lys Asp
Pro Asp Val Asn Met Leu His Val Phe Val 225 230
235 240 Leu Gly Glu Trp Gln Pro Ile Glu Tyr Gly Lys
Lys Lys Leu Lys Tyr 245 250
255 Leu Pro Tyr Asn His Gln His Glu Tyr Phe Phe Leu Ile Gly Pro Pro
260 265 270 Leu Leu
Ile Pro Met Tyr Phe Gln Tyr Gln Ile Ile Met Thr Met Ile 275
280 285 Val His Lys Asn Trp Val Asp
Leu Ala Trp Ala Val Ser Tyr Tyr Ile 290 295
300 Arg Phe Phe Ile Thr Tyr Ile Pro Phe Tyr Gly Ile
Leu Gly Ala Leu 305 310 315
320 Leu Phe Leu Asn Phe Ile Arg Phe Leu Glu Ser His Trp Phe Val Trp
325 330 335 Val Thr Gln
Met Asn His Ile Val Met Glu Ile Asp Gln Glu Ala Tyr 340
345 350 Arg Asp Trp Phe Ser Ser Gln Leu
Thr Ala Thr Cys Asn Val Glu Gln 355 360
365 Ser Phe Phe Asn Asp Trp Phe Ser Gly His Leu Asn Phe
Gln Ile Glu 370 375 380
His His Leu Phe Pro Thr Met Pro Arg His Asn Leu His Lys Ile Ala 385
390 395 400 Pro Leu Val Lys
Ser Leu Cys Ala Lys His Gly Ile Glu Tyr Gln Glu 405
410 415 Lys Pro Leu Leu Arg Ala Leu Leu Asp
Ile Ile Arg Ser Leu Lys Lys 420 425
430 Ser Gly Lys Leu Trp Leu Asp Ala Tyr Leu His Lys
435 440 2444PRTMus musculus 2Met Gly Lys
Gly Gly Asn Gln Gly Glu Gly Ser Thr Glu Arg Gln Ala 1 5
10 15 Pro Met Pro Thr Phe Arg Trp Glu
Glu Ile Gln Lys His Asn Leu Arg 20 25
30 Thr Asp Arg Trp Leu Val Ile Asp Arg Lys Val Tyr Asn
Val Thr Lys 35 40 45
Trp Ser Gln Arg His Pro Gly Gly His Arg Val Ile Gly His Tyr Ser 50
55 60 Gly Glu Asp Ala
Thr Asp Ala Phe Arg Ala Phe His Leu Asp Leu Asp 65 70
75 80 Phe Val Gly Lys Phe Leu Lys Pro Leu
Leu Ile Gly Glu Leu Ala Pro 85 90
95 Glu Glu Pro Ser Leu Asp Arg Gly Lys Ser Ser Gln Ile Thr
Glu Asp 100 105 110
Phe Arg Ala Leu Lys Lys Thr Ala Glu Asp Met Asn Leu Phe Lys Thr
115 120 125 Asn His Leu Phe
Phe Phe Leu Leu Leu Ser His Ile Ile Val Met Glu 130
135 140 Ser Leu Ala Trp Phe Ile Leu Ser
Tyr Phe Gly Thr Gly Trp Ile Pro 145 150
155 160 Thr Leu Val Thr Ala Phe Val Leu Ala Thr Ser Gln
Ala Gln Ala Gly 165 170
175 Trp Leu Gln His Asp Tyr Gly His Leu Ser Val Tyr Lys Lys Ser Ile
180 185 190 Trp Asn His
Val Val His Lys Phe Val Ile Gly His Leu Lys Gly Ala 195
200 205 Ser Ala Asn Trp Trp Asn His Arg
His Phe Gln His His Ala Lys Pro 210 215
220 Asn Ile Phe His Lys Asp Pro Asp Ile Lys Ser Leu His
Val Phe Val 225 230 235
240 Leu Gly Glu Trp Gln Pro Leu Glu Tyr Gly Lys Lys Lys Leu Lys Tyr
245 250 255 Leu Pro Tyr Asn
His Gln His Glu Tyr Phe Phe Leu Ile Gly Pro Pro 260
265 270 Leu Leu Ile Pro Met Tyr Phe Gln Tyr
Gln Ile Ile Met Thr Met Ile 275 280
285 Ser Arg Arg Asp Trp Val Asp Leu Ala Trp Ala Ile Ser Tyr
Tyr Met 290 295 300
Arg Phe Phe Tyr Thr Tyr Ile Pro Phe Tyr Gly Ile Leu Gly Ala Leu 305
310 315 320 Val Phe Leu Asn Phe
Ile Arg Phe Leu Glu Ser His Trp Phe Val Trp 325
330 335 Val Thr Gln Met Asn His Leu Val Met Glu
Ile Asp Leu Asp His Tyr 340 345
350 Arg Asp Trp Phe Ser Ser Gln Leu Ala Ala Thr Cys Asn Val Glu
Gln 355 360 365 Ser
Phe Phe Asn Asp Trp Phe Ser Gly His Leu Asn Phe Gln Ile Glu 370
375 380 His His Leu Phe Pro Thr
Met Pro Arg His Asn Leu His Lys Ile Ala 385 390
395 400 Pro Leu Val Lys Ser Leu Cys Ala Lys His Gly
Ile Glu Tyr Gln Glu 405 410
415 Lys Pro Leu Leu Arg Ala Leu Ile Asp Ile Val Ser Ser Leu Lys Lys
420 425 430 Ser Gly
Glu Leu Trp Leu Asp Ala Tyr Leu His Lys 435 440
3459PRTPythium irregulare 3Met Val Asp Leu Lys Pro Gly Val
Lys Arg Leu Val Ser Trp Lys Glu 1 5 10
15 Ile Arg Glu His Ala Thr Pro Ala Thr Ala Trp Ile Val
Ile His His 20 25 30
Lys Val Tyr Asp Ile Ser Lys Trp Asp Ser His Pro Gly Gly Ser Val
35 40 45 Met Leu Thr Gln
Ala Gly Glu Asp Ala Thr Asp Ala Phe Ala Val Phe 50
55 60 His Pro Ser Ser Ala Leu Lys Leu
Leu Glu Gln Phe Tyr Val Gly Asp 65 70
75 80 Val Asp Glu Thr Ser Lys Ala Glu Ile Glu Gly Glu
Pro Ala Ser Asp 85 90
95 Glu Glu Arg Ala Arg Arg Glu Arg Ile Asn Glu Phe Ile Ala Ser Tyr
100 105 110 Arg Arg Leu
Arg Val Lys Val Lys Gly Met Gly Leu Tyr Asp Ala Ser 115
120 125 Ala Leu Tyr Tyr Ala Trp Lys Leu
Val Ser Thr Phe Gly Ile Ala Val 130 135
140 Leu Ser Met Ala Ile Cys Phe Phe Phe Asn Ser Phe Ala
Met Tyr Met 145 150 155
160 Val Ala Gly Val Ile Met Gly Leu Phe Tyr Gln Gln Ser Gly Trp Leu
165 170 175 Ala His Asp Phe
Leu His Asn Gln Val Cys Glu Asn Arg Thr Leu Gly 180
185 190 Asn Leu Ile Gly Cys Leu Val Gly Asn
Ala Trp Gln Gly Phe Ser Val 195 200
205 Gln Trp Trp Lys Asn Lys His Asn Leu His His Ala Val Pro
Asn Leu 210 215 220
His Ser Ala Lys Asp Glu Gly Phe Ile Gly Asp Pro Asp Ile Asp Thr 225
230 235 240 Met Pro Leu Leu Ala
Trp Ser Lys Glu Met Ala Arg Lys Ala Phe Glu 245
250 255 Ser Ala His Gly Pro Phe Phe Ile Arg Asn
Gln Ala Phe Leu Tyr Phe 260 265
270 Pro Leu Leu Leu Leu Ala Arg Leu Ser Trp Leu Ala Gln Ser Phe
Phe 275 280 285 Tyr
Val Phe Thr Glu Phe Ser Phe Gly Ile Phe Asp Lys Val Glu Phe 290
295 300 Asp Gly Pro Glu Lys Ala
Gly Leu Ile Val His Tyr Ile Trp Gln Leu 305 310
315 320 Ala Ile Pro Tyr Phe Cys Asn Met Ser Leu Phe
Glu Gly Val Ala Tyr 325 330
335 Phe Leu Met Gly Gln Ala Ser Cys Gly Leu Leu Leu Ala Leu Val Phe
340 345 350 Ser Ile
Gly His Asn Gly Met Ser Val Tyr Glu Arg Glu Thr Lys Pro 355
360 365 Asp Phe Trp Gln Leu Gln Val
Thr Thr Thr Arg Asn Ile Arg Ala Ser 370 375
380 Val Phe Met Asp Trp Phe Thr Gly Gly Leu Asn Tyr
Gln Ile Asp His 385 390 395
400 His Leu Phe Pro Leu Val Pro Arg His Asn Leu Pro Lys Val Asn Val
405 410 415 Leu Ile Lys
Ser Leu Cys Lys Glu Phe Asp Ile Pro Phe His Glu Thr 420
425 430 Gly Phe Trp Glu Gly Ile Tyr Glu
Val Val Asp His Leu Ala Asp Ile 435 440
445 Ser Lys Glu Phe Ile Thr Glu Phe Pro Ala Met 450
455 4448PRTBorago officinalis 4Met Ala
Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn 1 5
10 15 His Asp Lys Pro Gly Asp Leu
Trp Ile Ser Ile Gln Gly Lys Ala Tyr 20 25
30 Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly
Ser Phe Pro Leu 35 40 45
Lys Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His
50 55 60 Pro Ala Ser
Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr 65
70 75 80 Leu Lys Asp Tyr Ser Val Ser
Glu Val Ser Lys Asp Tyr Arg Lys Leu 85
90 95 Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp
Lys Lys Gly His Ile 100 105
110 Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met Ser
Val 115 120 125 Tyr
Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Phe Ser Gly 130
135 140 Cys Leu Met Gly Phe Leu
Trp Ile Gln Ser Gly Trp Ile Gly His Asp 145 150
155 160 Ala Gly His Tyr Met Val Val Ser Asp Ser Arg
Leu Asn Lys Phe Met 165 170
175 Gly Ile Phe Ala Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp
180 185 190 Lys Trp
Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr 195
200 205 Asp Pro Asp Leu Gln Tyr Ile
Pro Phe Leu Val Val Ser Ser Lys Phe 210 215
220 Phe Gly Ser Leu Thr Ser His Phe Tyr Glu Lys Arg
Leu Thr Phe Asp 225 230 235
240 Ser Leu Ser Arg Phe Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro
245 250 255 Ile Met Cys
Ala Ala Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met 260
265 270 Leu Leu Thr Lys Arg Asn Val Ser
Tyr Arg Ala Gln Glu Leu Leu Gly 275 280
285 Cys Leu Val Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser
Cys Leu Pro 290 295 300
Asn Trp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr 305
310 315 320 Gly Met Gln Gln
Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val 325
330 335 Tyr Val Gly Lys Pro Lys Gly Asn Asn
Trp Phe Glu Lys Gln Thr Asp 340 345
350 Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe
His Gly 355 360 365
Gly Leu Gln Phe Gln Ile Glu His His Leu Phe Pro Lys Met Pro Arg 370
375 380 Cys Asn Leu Arg Lys
Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys 385 390
395 400 His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe
Ser Lys Ala Asn Glu Met 405 410
415 Thr Leu Arg Thr Leu Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile
Thr 420 425 430 Lys
Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu His Thr His Gly 435
440 445 5446PRTAnemone
leveillei 5Met Ala Glu Lys Arg Arg Ser Ile Ser Ser Asp Asp Leu Arg Ser
His 1 5 10 15 Asn
Lys Pro Gly Asp Val Trp Ile Ser Ile Gln Gly Lys Ile Tyr Asp
20 25 30 Val Thr Glu Trp Gly
Lys Asp His Pro Gly Gly Glu Gly Pro Leu Leu 35
40 45 Asn Leu Ala Gly Gln Asp Val Thr Asp
Ala Phe Val Ala Phe His Pro 50 55
60 Gly Ser Ala Trp Lys Asn Leu Asp Lys Phe His Ile Gly
Tyr Leu Gln 65 70 75
80 Asp Tyr Val Val Ser Asp Val Ser Lys Asp Tyr Arg Lys Leu Val Ser
85 90 95 Glu Phe Ser Lys
Ala Gly Leu Tyr Glu Lys Lys Gly His Gly His Leu 100
105 110 Ile Arg Leu Leu Val Met Ser Leu Val
Phe Ile Ala Ser Val Ser Gly 115 120
125 Val Val Leu Ser Asp Lys Thr Ser Val His Val Gly Ser Ala
Val Leu 130 135 140
Leu Ala Val Ile Trp Met Gln Phe Gly Phe Ile Gly His Asp Ser Gly 145
150 155 160 His Tyr Asn Ile Met
Thr Ser Pro Glu Leu Asn Arg Tyr Met Gln Ile 165
170 175 Phe Ser Val Asn Val Val Ser Gly Val Ser
Val Gly Trp Trp Lys Arg 180 185
190 Tyr His Asn Ala His His Ile Ala Val Asn Ser Leu Glu Tyr Asp
Pro 195 200 205 Asp
Leu Gln Tyr Val Pro Phe Leu Val Val Ser Thr Ala Ile Phe Asp 210
215 220 Ser Leu Thr Ser His Phe
Tyr Arg Lys Lys Met Thr Phe Asp Ala Val 225 230
235 240 Ala Arg Phe Leu Val Ser Phe Gln His Trp Thr
Phe Tyr Pro Leu Met 245 250
255 Ala Ile Gly Arg Val Ser Phe Leu Ala Gln Ser Ile Gly Val Leu Leu
260 265 270 Ser Lys
Lys Pro Leu Pro Asp Arg His Leu Glu Trp Phe Gly Leu Val 275
280 285 Val Phe Trp Ala Trp Tyr Ser
Leu Leu Ile Ser Cys Leu Pro Asn Trp 290 295
300 Trp Glu Arg Val Ile Phe Ile Ala Val Asn Phe Ala
Val Thr Gly Ile 305 310 315
320 Gln His Val Gln Phe Cys Leu Asn His Tyr Ser Ala Gln Thr Tyr Ile
325 330 335 Gly Ala Pro
Cys Ala Asn Asp Trp Phe Glu Lys Gln Thr Lys Gly Ser 340
345 350 Ile Asp Ile Ser Cys Ser Pro Trp
Thr Asp Trp Phe His Gly Gly Leu 355 360
365 Gln Phe Gln Ile Glu His His Leu Phe Pro Arg Met Pro
Arg Cys Asn 370 375 380
Leu Arg Lys Ile Ser Pro Phe Val Lys Glu Leu Cys Arg Lys His Asn 385
390 395 400 Leu Val Tyr Thr
Ser Val Ser Phe Phe Glu Gly Asn Arg Arg Thr Leu 405
410 415 Ala Thr Leu Lys Asn Ala Ala Leu Lys
Ala Arg Asp Leu Thr Ser Pro 420 425
430 Ile Pro Lys Asn Leu Val Trp Glu Ala Val His Thr His Gly
435 440 445 6520PRTCeratodon
purpureus 6Met Val Ser Gln Gly Gly Gly Leu Ser Gln Gly Ser Ile Glu Glu
Asn 1 5 10 15 Ile
Asp Val Glu His Leu Ala Thr Met Pro Leu Val Ser Asp Phe Leu
20 25 30 Asn Val Leu Gly Thr
Thr Leu Gly Gln Trp Ser Leu Ser Thr Thr Phe 35
40 45 Ala Phe Lys Arg Leu Thr Thr Lys Lys
His Ser Ser Asp Ile Ser Val 50 55
60 Glu Ala Gln Lys Glu Ser Val Ala Arg Gly Pro Val Glu
Asn Ile Ser 65 70 75
80 Gln Ser Val Ala Gln Pro Ile Arg Arg Arg Trp Val Gln Asp Lys Lys
85 90 95 Pro Val Thr Tyr
Ser Leu Lys Asp Val Ala Ser His Asp Met Pro Gln 100
105 110 Asp Cys Trp Ile Ile Ile Lys Glu Lys
Val Tyr Asp Val Ser Thr Phe 115 120
125 Ala Glu Gln His Pro Gly Gly Thr Val Ile Asn Thr Tyr Phe
Gly Arg 130 135 140
Asp Ala Thr Asp Val Phe Ser Thr Phe His Ala Ser Thr Ser Trp Lys 145
150 155 160 Ile Leu Gln Asn Phe
Tyr Ile Gly Asn Leu Val Arg Glu Glu Pro Thr 165
170 175 Leu Glu Leu Leu Lys Glu Tyr Arg Glu Leu
Arg Ala Leu Phe Leu Arg 180 185
190 Glu Gln Leu Phe Lys Ser Ser Lys Ser Tyr Tyr Leu Phe Lys Thr
Leu 195 200 205 Ile
Asn Val Ser Ile Val Ala Thr Ser Ile Ala Ile Ile Ser Leu Tyr 210
215 220 Lys Ser Tyr Arg Ala Val
Leu Leu Ser Ala Ser Leu Met Gly Leu Phe 225 230
235 240 Ile Gln Gln Cys Gly Trp Leu Ser His Asp Phe
Leu His His Gln Val 245 250
255 Phe Glu Thr Arg Trp Leu Asn Asp Val Val Gly Tyr Val Val Gly Asn
260 265 270 Val Val
Leu Gly Phe Ser Val Ser Trp Trp Lys Thr Lys His Asn Leu 275
280 285 His His Ala Ala Pro Asn Glu
Cys Asp Gln Lys Tyr Thr Pro Ile Asp 290 295
300 Glu Asp Ile Asp Thr Leu Pro Ile Ile Ala Trp Ser
Lys Asp Leu Leu 305 310 315
320 Ala Thr Val Glu Ser Lys Thr Met Leu Arg Val Leu Gln Tyr Gln His
325 330 335 Leu Phe Phe
Leu Val Leu Leu Thr Phe Ala Arg Ala Ser Trp Leu Phe 340
345 350 Trp Ser Ala Ala Phe Thr Leu Arg
Pro Glu Leu Thr Leu Gly Glu Lys 355 360
365 Leu Leu Glu Arg Gly Thr Met Ala Leu His Tyr Ile Trp
Phe Asn Ser 370 375 380
Val Ala Phe Tyr Leu Leu Pro Gly Trp Lys Pro Val Val Trp Met Val 385
390 395 400 Val Ser Glu Leu
Met Ser Gly Phe Leu Leu Gly Tyr Val Phe Val Leu 405
410 415 Ser His Asn Gly Met Glu Val Tyr Asn
Thr Ser Lys Asp Phe Val Asn 420 425
430 Ala Gln Ile Ala Ser Thr Arg Asp Ile Lys Ala Gly Val Phe
Asn Asp 435 440 445
Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu His His Leu Phe Pro 450
455 460 Thr Met Pro Arg His
Asn Leu Asn Lys Ile Ser Pro His Val Glu Thr 465 470
475 480 Leu Cys Lys Lys His Gly Leu Val Tyr Glu
Asp Val Ser Met Ala Ser 485 490
495 Gly Thr Tyr Arg Val Leu Lys Thr Leu Lys Asp Val Ala Asp Ala
Ala 500 505 510 Ser
His Gln Gln Leu Ala Ala Ser 515 520
7525PRTPhyscomitrella patens 7Met 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 8457PRTMortierella alpina 8Met Ala Ala Ala
Pro Ser Val Arg Thr Phe Thr Arg Ala Glu Ile Leu 1 5
10 15 Asn Ala Glu Ala Leu Asn Glu Gly Lys
Lys Asp Ala Glu Ala Pro Phe 20 25
30 Leu Met Ile Ile Asp Asn Lys Val Tyr Asp Val Arg Glu Phe
Val Pro 35 40 45
Asp His Pro Gly Gly Ser Val Ile Leu Thr His Val Gly Lys Asp Gly 50
55 60 Thr Asp Val Phe Asp
Thr Phe His Pro Glu Ala Ala Trp Glu Thr Leu 65 70
75 80 Ala Asn Phe Tyr Val Gly Asp Ile Asp Glu
Ser Asp Arg Ala Ile Lys 85 90
95 Asn Asp Asp Phe Ala Ala Glu Val Arg Lys Leu Arg Thr Leu Phe
Gln 100 105 110 Ser
Leu Gly Tyr Tyr Asp Ser Ser Lys Ala Tyr Tyr Ala Phe Lys Val 115
120 125 Ser Phe Asn Leu Cys Ile
Trp Gly Leu Ser Thr Phe Ile Val Ala Lys 130 135
140 Trp Gly Gln Thr Ser Thr Leu Ala Asn Val Leu
Ser Ala Ala Leu Leu 145 150 155
160 Gly Leu Phe Trp Gln Gln Cys Gly Trp Leu Ala His Asp Phe Leu His
165 170 175 His Gln
Val Phe Gln Asp Arg Phe Trp Gly Asp Leu Phe Gly Ala Phe 180
185 190 Leu Gly Gly Val Cys Gln Gly
Phe Ser Ser Ser Trp Trp Lys Asp Lys 195 200
205 His Asn Thr His His Ala Ala Pro Asn Val His Gly
Glu Asp Pro Asp 210 215 220
Ile Asp Thr His Pro Leu Leu Thr Trp Ser Glu His Ala Leu Glu Met 225
230 235 240 Phe Ser Asp
Val Pro Asp Glu Glu Leu Thr Arg Met Trp Ser Arg Phe 245
250 255 Met Val Leu Asn Gln Thr Trp Phe
Tyr Phe Pro Ile Leu Ser Phe Ala 260 265
270 Arg Leu Ser Trp Cys Leu Gln Ser Ile Met Phe Val Leu
Pro Asn Gly 275 280 285
Gln Ala His Lys Pro Ser Gly Ala Arg Val Pro Ile Ser Leu Val Glu 290
295 300 Gln Leu Ser Leu
Ala Met His Trp Thr Trp Tyr Leu Ala Thr Met Phe 305 310
315 320 Leu Phe Ile Lys Asp Pro Val Asn Met
Ile Val Tyr Phe Leu Val Ser 325 330
335 Gln Ala Val Cys Gly Asn Leu Leu Ala Ile Val Phe Ser Leu
Asn His 340 345 350
Asn Gly Met Pro Val Ile Ser Lys Glu Glu Ala Val Asp Met Asp Phe
355 360 365 Phe Thr Lys Gln
Ile Ile Thr Gly Arg Asp Val His Pro Gly Leu Phe 370
375 380 Ala Asn Trp Phe Thr Gly Gly Leu
Asn Tyr Gln Ile Glu His His Leu 385 390
395 400 Phe Pro Ser Met Pro Arg His Asn Phe Ser Lys Ile
Gln Pro Ala Val 405 410
415 Glu Thr Leu Cys Lys Lys Tyr Gly Val Arg Tyr His Thr Thr Gly Met
420 425 430 Ile Glu Gly
Thr Ala Glu Val Phe Ser Arg Leu Asn Glu Val Ser Lys 435
440 445 Ala Ala Ser Lys Met Gly Lys Ala
Gln 450 455 9443PRTCaenorhabditis elegans
9Met Val Val Asp Lys Asn Ala Ser Gly Leu Arg Met Lys Val Asp Gly 1
5 10 15 Lys Trp Leu Tyr
Leu Ser Glu Glu Leu Val Lys Lys His Pro Gly Gly 20
25 30 Ala Val Ile Glu Gln Tyr Arg Asn Ser
Asp Ala Thr His Ile Phe His 35 40
45 Ala Phe His Glu Gly Ser Ser Gln Ala Tyr Lys Gln Leu Asp
Leu Leu 50 55 60
Lys Lys His Gly Glu His Asp Glu Phe Leu Glu Lys Gln Leu Glu Lys 65
70 75 80 Arg Leu Asp Lys Val
Asp Ile Asn Val Ser Ala Tyr Asp Val Ser Val 85
90 95 Ala Gln Glu Lys Lys Met Val Glu Ser Phe
Glu Lys Leu Arg Gln Lys 100 105
110 Leu His Asp Asp Gly Leu Met Lys Ala Asn Glu Thr Tyr Phe Leu
Phe 115 120 125 Lys
Ala Ile Ser Thr Leu Ser Ile Met Ala Phe Ala Phe Tyr Leu Gln 130
135 140 Tyr Leu Gly Trp Tyr Ile
Thr Ser Ala Cys Leu Leu Ala Leu Ala Trp 145 150
155 160 Gln Gln Phe Gly Trp Leu Thr His Glu Phe Cys
His Gln Gln Pro Thr 165 170
175 Lys Asn Arg Pro Leu Asn Asp Thr Ile Ser Leu Phe Phe Gly Asn Phe
180 185 190 Leu Gln
Gly Phe Ser Arg Asp Trp Trp Lys Asp Lys His Asn Thr His 195
200 205 His Ala Ala Thr Asn Val Ile
Asp His Asp Gly Asp Ile Asp Leu Ala 210 215
220 Pro Leu Phe Ala Phe Ile Pro Gly Asp Leu Cys Lys
Tyr Lys Ala Ser 225 230 235
240 Phe Glu Lys Ala Ile Leu Lys Ile Val Pro Tyr Gln His Leu Tyr Phe
245 250 255 Thr Ala Met
Leu Pro Met Leu Arg Phe Ser Trp Thr Gly Gln Ser Val 260
265 270 Gln Trp Val Phe Lys Glu Asn Gln
Met Glu Tyr Lys Val Tyr Gln Arg 275 280
285 Asn Ala Phe Trp Glu Gln Ala Thr Ile Val Gly His Trp
Ala Trp Val 290 295 300
Phe Tyr Gln Leu Phe Leu Leu Pro Thr Trp Pro Leu Arg Val Ala Tyr 305
310 315 320 Phe Ile Ile Ser
Gln Met Gly Gly Gly Leu Leu Ile Ala His Val Val 325
330 335 Thr Phe Asn His Asn Ser Val Asp Lys
Tyr Pro Ala Asn Ser Arg Ile 340 345
350 Leu Asn Asn Phe Ala Ala Leu Gln Ile Leu Thr Thr Arg Asn
Met Thr 355 360 365
Pro Ser Pro Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln Ile 370
375 380 Glu His His Leu Phe
Pro Thr Met Pro Arg Cys Asn Leu Asn Ala Cys 385 390
395 400 Val Lys Tyr Val Lys Glu Trp Cys Lys Glu
Asn Asn Leu Pro Tyr Leu 405 410
415 Val Asp Asp Tyr Phe Asp Gly Tyr Ala Met Asn Leu Gln Gln Leu
Lys 420 425 430 Asn
Met Ala Glu His Ile Gln Ala Lys Ala Ala 435 440
10448PRTEchium plantagineum 10Met Ala Asn Ala Ile Lys Lys Tyr
Ile Thr Ala Glu Glu Leu Lys Lys 1 5 10
15 His Asp Lys Ala Gly Asp Leu Trp Ile Ser Ile Gln Gly
Lys Ile Tyr 20 25 30
Asp Val Ser Asp Trp Leu Lys Asp His Pro Gly Gly Asn Phe Pro Leu
35 40 45 Leu Ser Leu Ala
Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His 50
55 60 Ser Gly Thr Thr Trp Lys Leu Leu
Glu Lys Phe Phe Thr Gly Tyr Tyr 65 70
75 80 Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp
Tyr Arg Lys Leu 85 90
95 Val Phe Glu Phe Asn Lys Met Gly Leu Phe Asp Lys Lys Gly His Ile
100 105 110 Val Leu Val
Thr Val Leu Phe Ile Ala Met Leu Phe Gly Met Ser Val 115
120 125 Tyr Gly Val Leu Phe Cys Glu Gly
Val Leu Val His Leu Leu Ala Gly 130 135
140 Gly Leu Met Gly Phe Val Trp Ile Gln Ser Gly Trp Ile
Gly His Asp 145 150 155
160 Ala Gly His Tyr Ile Val Met Pro Asp Ala Arg Leu Asn Lys Leu Met
165 170 175 Gly Ile Val Ala
Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp 180
185 190 Lys Trp Asn His Asn Ala His His Ile
Ala Cys Asn Ser Leu Asp Tyr 195 200
205 Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser
Lys Leu 210 215 220
Phe Ser Ser Leu Thr Ser His Phe Tyr Glu Lys Lys Leu Thr Phe Asp 225
230 235 240 Ser Leu Ser Arg Phe
Phe Val Ser His Gln His Trp Thr Phe Tyr Pro 245
250 255 Val Met Cys Met Ala Arg Val Asn Met Phe
Val Gln Ser Leu Ile Met 260 265
270 Leu Leu Thr Lys Arg Asn Val Phe Tyr Arg Ser Gln Glu Leu Leu
Gly 275 280 285 Leu
Val Val Phe Trp Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro 290
295 300 Asn Trp Gly Glu Arg Val
Met Phe Val Val Ala Ser Leu Ser Val Thr 305 310
315 320 Gly Met Gln Gln Val Gln Phe Ser Leu Asn His
Phe Ser Ser Ser Val 325 330
335 Tyr Val Gly Gln Pro Lys Gly Asn Asp Trp Phe Glu Lys Gln Thr Cys
340 345 350 Gly Thr
Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly 355
360 365 Gly Leu Gln Phe Gln Val Glu
His His Leu Phe Pro Lys Leu Pro Arg 370 375
380 Cys His Leu Arg Lys Ile Ser Pro Phe Val Met Glu
Leu Cys Lys Lys 385 390 395
400 His Asn Leu Ser Tyr Asn Cys Ala Ser Phe Ser Glu Ala Asn Asn Met
405 410 415 Thr Leu Arg
Thr Leu Arg Asp Thr Ala Leu Gln Ala Arg Asp Leu Thr 420
425 430 Lys Pro Leu Pro Lys Asn Leu Val
Trp Glu Ala Leu Asn Thr His Gly 435 440
445 11448PRTEchium gentianoides 11Met Ala Asn Ala Ile
Lys Lys Tyr Ile Thr Ala Glu Glu Leu Lys Lys 1 5
10 15 His Asp Lys Glu Gly Asp Leu Trp Ile Ser
Ile Gln Gly Lys Val Tyr 20 25
30 Asp Val Ser Asp Trp Leu Lys Asp His Pro Gly Gly Lys Phe Pro
Leu 35 40 45 Leu
Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His 50
55 60 Ser Gly Ser Thr Trp Lys
Phe Leu Asp Ser Phe Phe Thr Gly Tyr Tyr 65 70
75 80 Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys
Asp Tyr Arg Lys Leu 85 90
95 Val Phe Glu Phe Asn Lys Met Gly Leu Phe Asp Lys Lys Gly His Ile
100 105 110 Val Leu
Val Thr Val Leu Phe Ile Ala Met Met Phe Ala Met Ser Val 115
120 125 Tyr Gly Val Leu Phe Cys Glu
Gly Val Leu Val His Leu Leu Ala Gly 130 135
140 Gly Leu Met Gly Phe Val Trp Ile Gln Ser Gly Trp
Ile Gly His Asp 145 150 155
160 Ala Gly His Tyr Ile Val Met Pro Asn Pro Arg Leu Asn Lys Leu Met
165 170 175 Gly Ile Val
Ala Gly Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp 180
185 190 Lys Trp Asn His Asn Ala His His
Ile Ala Cys Asn Ser Leu Asp Tyr 195 200
205 Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser
Ser Lys Leu 210 215 220
Phe Ser Ser Leu Thr Ser His Phe Tyr Glu Lys Lys Leu Thr Phe Asp 225
230 235 240 Ser Leu Ser Arg
Phe Phe Val Ser His Gln His Trp Thr Phe Tyr Pro 245
250 255 Val Met Cys Ser Ala Arg Val Asn Met
Phe Val Gln Ser Leu Ile Met 260 265
270 Leu Leu Thr Lys Arg Asn Val Phe Tyr Arg Ser Gln Glu Leu
Leu Gly 275 280 285
Leu Val Val Phe Trp Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro 290
295 300 Asn Trp Gly Glu Arg
Ile Met Phe Val Val Ala Ser Leu Ser Val Thr 305 310
315 320 Gly Met Gln Gln Val Gln Phe Ser Leu Asn
His Phe Ser Ala Ser Val 325 330
335 Tyr Val Gly Gln Pro Lys Gly Asn Asp Trp Phe Glu Lys Gln Thr
Cys 340 345 350 Gly
Thr Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly 355
360 365 Gly Leu Gln Phe Gln Val
Glu His His Leu Phe Pro Lys Leu Pro Arg 370 375
380 Cys His Leu Arg Lys Ile Ser Pro Phe Val Met
Glu Leu Cys Lys Lys 385 390 395
400 His Asn Leu Ser Tyr Asn Cys Ala Ser Phe Ser Glu Ala Asn Glu Met
405 410 415 Thr Leu
Arg Thr Leu Arg Asp Thr Ala Leu Gln Ala Arg Asp Leu Thr 420
425 430 Lys Pro Leu Pro Lys Asn Leu
Val Trp Glu Ala Leu Asn Thr His Gly 435 440
445 12448PRTEchium pitardii 12Met Ala Asn Ala Ile
Lys Lys Tyr Ile Thr Ala Glu Glu Leu Lys Lys 1 5
10 15 His Asp Lys Glu Gly Asp Leu Trp Ile Ser
Ile Gln Gly Lys Val Tyr 20 25
30 Asp Val Ser Asp Trp Leu Lys Asp His Pro Gly Gly Lys Phe Pro
Leu 35 40 45 Leu
Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His 50
55 60 Ser Gly Ser Thr Trp Lys
Leu Leu Asp Ser Phe Phe Thr Gly Tyr Tyr 65 70
75 80 Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys
Asp Tyr Arg Lys Leu 85 90
95 Val Phe Glu Phe Asn Lys Met Gly Leu Phe Asp Lys Lys Gly His Ile
100 105 110 Val Leu
Val Thr Val Phe Phe Ile Ala Met Met Phe Ala Met Ser Val 115
120 125 Tyr Gly Val Leu Phe Cys Glu
Gly Val Leu Val His Leu Leu Ala Gly 130 135
140 Gly Leu Met Gly Phe Val Trp Ile Gln Ser Gly Trp
Ile Gly His Asp 145 150 155
160 Ala Gly His Tyr Ile Val Met Pro Asn Pro Lys Leu Asn Lys Leu Met
165 170 175 Gly Ile Val
Ala Ser Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp 180
185 190 Lys Trp Asn His Asn Ala His His
Ile Ala Cys Asn Ser Leu Asp Tyr 195 200
205 Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser
Ser Lys Leu 210 215 220
Phe Ser Ser Leu Thr Ser His Phe Tyr Glu Lys Lys Leu Thr Phe Asp 225
230 235 240 Ser Leu Ser Arg
Phe Phe Val Ser His Gln His Trp Thr Phe Tyr Pro 245
250 255 Val Met Cys Ser Ala Arg Val Asn Met
Phe Val Gln Ser Leu Ile Met 260 265
270 Leu Leu Thr Lys Arg Asn Val Phe Tyr Arg Ser Gln Glu Leu
Leu Gly 275 280 285
Leu Val Val Phe Trp Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro 290
295 300 Asn Trp Gly Glu Arg
Ile Met Phe Val Val Ala Ser Leu Ser Val Thr 305 310
315 320 Gly Leu Gln Gln Val Gln Phe Ser Leu Asn
His Phe Ala Ala Ser Val 325 330
335 Tyr Val Gly Gln Pro Lys Gly Ile Asp Trp Phe Glu Lys Gln Thr
Cys 340 345 350 Gly
Thr Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly 355
360 365 Gly Leu Gln Phe Gln Val
Glu His His Leu Phe Pro Lys Leu Pro Arg 370 375
380 Cys His Leu Arg Lys Ile Ser Pro Phe Val Met
Glu Leu Cys Lys Lys 385 390 395
400 His Asn Leu Ser Tyr Asn Cys Ala Ser Phe Ser Gln Ala Asn Glu Met
405 410 415 Thr Leu
Arg Thr Leu Arg Asp Thr Ala Leu Gln Ala Arg Asp Leu Thr 420
425 430 Lys Pro Leu Pro Lys Asn Leu
Val Trp Glu Ala Leu Asn Thr His Gly 435 440
445 13444PRTDanio rerio 13Met Gly Gly Gly Gly Gln
Gln Thr Asp Arg Ile Thr Asp Thr Asn Gly 1 5
10 15 Arg Phe Ser Ser Tyr Thr Trp Glu Glu Val Gln
Lys His Thr Lys His 20 25
30 Gly Asp Gln Trp Val Val Val Glu Arg Lys Val Tyr Asn Val Ser
Gln 35 40 45 Trp
Val Lys Arg His Pro Gly Gly Leu Arg Ile Leu Gly His Tyr Ala 50
55 60 Gly Glu Asp Ala Thr Glu
Ala Phe Thr Ala Phe His Pro Asn Leu Gln 65 70
75 80 Leu Val Arg Lys Tyr Leu Lys Pro Leu Leu Ile
Gly Glu Leu Glu Ala 85 90
95 Ser Glu Pro Ser Gln Asp Arg Gln Lys Asn Ala Ala Leu Val Glu Asp
100 105 110 Phe Arg
Ala Leu Arg Glu Arg Leu Glu Ala Glu Gly Cys Phe Lys Thr 115
120 125 Gln Pro Leu Phe Phe Ala Leu
His Leu Gly His Ile Leu Leu Leu Glu 130 135
140 Ala Ile Ala Phe Met Met Val Trp Tyr Phe Gly Thr
Gly Trp Ile Asn 145 150 155
160 Thr Leu Ile Val Ala Val Ile Leu Ala Thr Ala Gln Ser Gln Ala Gly
165 170 175 Trp Leu Gln
His Asp Phe Gly His Leu Ser Val Phe Lys Thr Ser Gly 180
185 190 Met Asn His Leu Val His Lys Phe
Val Ile Gly His Leu Lys Gly Ala 195 200
205 Ser Ala Gly Trp Trp Asn His Arg His Phe Gln His His
Ala Lys Pro 210 215 220
Asn Ile Phe Lys Lys Asp Pro Asp Val Asn Met Leu Asn Ala Phe Val 225
230 235 240 Val Gly Asn Val
Gln Pro Val Glu Tyr Gly Val Lys Lys Ile Lys His 245
250 255 Leu Pro Tyr Asn His Gln His Lys Tyr
Phe Phe Phe Ile Gly Pro Pro 260 265
270 Leu Leu Ile Pro Val Tyr Phe Gln Phe Gln Ile Phe His Asn
Met Ile 275 280 285
Ser His Gly Met Trp Val Asp Leu Leu Trp Cys Ile Ser Tyr Tyr Val 290
295 300 Arg Tyr Phe Leu Cys
Tyr Thr Gln Phe Tyr Gly Val Phe Trp Ala Ile 305 310
315 320 Ile Leu Phe Asn Phe Val Arg Phe Met Glu
Ser His Trp Phe Val Trp 325 330
335 Val Thr Gln Met Ser His Ile Pro Met Asn Ile Asp Tyr Glu Lys
Asn 340 345 350 Gln
Asp Trp Leu Ser Met Gln Leu Val Ala Thr Cys Asn Ile Glu Gln 355
360 365 Ser Ala Phe Asn Asp Trp
Phe Ser Gly His Leu Asn Phe Gln Ile Glu 370 375
380 His His Leu Phe Pro Thr Val Pro Arg His Asn
Tyr Trp Arg Ala Ala 385 390 395
400 Pro Arg Val Arg Ala Leu Cys Glu Lys Tyr Gly Val Lys Tyr Gln Glu
405 410 415 Lys Thr
Leu Tyr Gly Ala Phe Ala Asp Ile Ile Arg Ser Leu Glu Lys 420
425 430 Ser Gly Glu Leu Trp Leu Asp
Ala Tyr Leu Asn Lys 435 440
148PRTArtificialConserved motif of d6 desaturases 14Met Ala Asn Ala Ile
Lys Lys Tyr 1 5 157PRTArtificialConserved
motif of d6 desaturases 15Glu Ala Leu Asn Thr His Gly 1 5
164PRTArtificialConserved motif of d6 desaturases 16His Pro Gly
Gly 1 1729DNAArtificialOligonucleotide primer 17actcgagcca
ccatggctaa tgcaatcaa
291828DNAArtificialOligonucleotide primer 18cctcgagctc aaccatgagt
attaagag
281921DNAArtificialOligonucleotide primer 19actctgtttc tgaggtgtcc a
212024DNAArtificialOligonucleotide primer 20catattaacc ctagccatac acat
242122DNAArtificialOligonucleotide primer 21ctccagcgat ggttgttgct at
222220DNAArtificialOligonucleotide primer 22aatgtctctg gtgacgtagc
20232070DNALinum usitatissimum
23ctagactcaa gcatacggac aagggtaaat aacatagtca ccagaacata ataaacaaaa
60agtgcagaag caagactaaa aaaattagct atggacattc aggttcatat tggaaacatc
120attatcctag tcttgtgacc atccttcctc ctgctctagt tgagaggcct tgggactaac
180gagaggtcag ttgggatagc agatccttat cctggactag cctttctggt gtttcagagt
240cttcgtgccg ccgtctacat ctatctccat taggtctgaa gatgactctt cacaccaacg
300acgtttaagg tctctatcct actcctagct tgcaatacct ggcttgcaat acctggagca
360tcgtgcacga tgattggata ctgtggagga ggagtgtttg ctgatttaga gctcccggtt
420gggtgatttg acttcgattt cagtttaggc ttgttgaaat ttttcaggtt ccattgtgaa
480gcctttagag cttgagcttc cttccatgtt aatgccttga tcgaattctc ctagagaaaa
540gggaagtcga tctctgagta ttgaaatcga agtgcacatt ttttttcaac gtgtccaatc
600aatccacaaa caaagcagaa gacaggtaat ctttcatact tatactgaca agtaatagtc
660ttaccgtcat gcataataac gtctcgttcc ttcaagaggg gttttccgac atccataacg
720acccgaagcc tcatgaaagc attagggaag aacttttggt tcttcttgtc atggccttta
780taggtgtcag ccgagctcgc caattcccgt ccgactggct ccgcaaaata ttcgaacggc
840aagttatgga cttgcaacca taactccacg gtattgagca ggacctattg tgaagactca
900tctcatggag cttcagaatg tggttgtcag caaaccaatg accgaaatcc atcacatgac
960ggacgtccag tgggtgagcg aaacgaaaca ggaagcgcct atctttcaga gtcgtgagct
1020ccacaccgga ttccggcaac tacgtgttgg gcaggcttcg ccgtattaga gatatgttga
1080ggcagaccca tctgtgccac tcgtacaatt acgagagttg ttttttttgt gattttccta
1140gtttctcgtt gatggtgagc tcatattcta catcgtatgg tctctcaacg tcgtttcctg
1200tcatctgata tcccgtcatt tgcatccacg tgcgccgcct cccgtgccaa gtccctaggt
1260gtcatgcacg ccaaattggt ggtggtgcgg gctgccctgt gcttcttacc gatgggtgga
1320ggttgagttt gggggtctcc gcggcgatgg tagtgggttg acggtttggt gtgggttgac
1380ggcattgatc aatttacttc ttgcttcaaa ttctttggca gaaaacaatt cattagatta
1440gaactggaaa ccagagtgat gagacggatt aagtcagatt ccaacagagt tacatctctt
1500aagaaataat gtaacccctt tagactttat atatttgcaa ttaaaaaaat aatttaactt
1560ttagacttta tatatagttt taataactaa gtttaaccac tctattattt atatcgaaac
1620tatttgtatg tctcccctct aaataaactt ggtattgtgt ttacagaacc tataatcaaa
1680taatcaatac tcaactgaag tttgtgcagt taattgaagg gattaacggc caaaatgcac
1740tagtattatc aaccgaatag attcacacta gatggccatt tccatcaata tcatcgccgt
1800tcttcttctg tccacatatc ccctctgaaa cttgagagac acctgcactt cattgtcctt
1860attacgtgtt acaaaatgaa acccatgcat ccatgcaaac tgaagaatgg cgcaagaacc
1920cttcccctcc atttcttatg tggcgaccat ccatttcacc atctcccgct ataaaacacc
1980cccatcactt cacctagaac atcatcacta cttgcttatc catccaaaag atacccacca
2040tggatccctg cagtaaatcc cgggctcgag
207024476DNALinum usitatissimum 24ctcgagcaag cttatgtgac gtgaaataat
aacggtaaaa tatatgtaat aataataata 60ataaagccac aaagtgagaa tgaggggaag
gggaaatgtg taatgagcca gtagccggtg 120gtgctaattt tgtatcgtat tgtcaataaa
tcatgaattt tgtggttttt atgtgttttt 180ttaaatcatg aattttaaat tttataaaat
aatctccaat cggaagaaca acattccata 240tccatgcatg gatgtttctt tacccaaatc
tagttcttga gaggcgttcc aaagatccca 300aacgaaacat attatctata ctaatactat
attattaatt actactgccc ggaatcacaa 360tccctgaatg attcctatta actacaagcc
ttgttggcgg cggagaagtg atcggcgcgg 420cgagaagcag cggactcgga gacgaggcct
tggaagatct gagtcgacac gggccc 476251425DNAEchium plantagineum
25atgaagcatc accgaacagt tctgcaacta tccctcaaaa gctttaaaat gaacaacaag
60gaacagagca accaccatgg ctaatgcaat caagaagtac attactgcag aggagctgaa
120gaagcatgat aaagcagggg atctctggat ctccattcaa ggaaaaatct atgatgtttc
180agattggttg aaggaccatc caggtgggaa cttccccttg ctgagccttg ctggccaaga
240ggtaactgat gcatttgttg catttcattc tggtacaact tggaagcttc ttgaaaaatt
300cttcactggt tattacctta aagattactc tgtttctgag gtgtccaaag attacaggaa
360gcttgtgttt gagtttaata aaatgggctt gtttgacaaa aagggtcata ttgttcttgt
420gactgtcttg tttatagcta tgttgtttgg tatgagtgtt tatggggttt tgttttgtga
480gggtgttttg gtacatttgc ttgctggggg gttgatgggt tttgtctgga ttcagagtgg
540ttggattggt catgatgctg ggcattatat tgttatgcct gatgctaggc ttaataagct
600tatgggtatt gttgctgcca attgtttatc tggaataagc attggttggt ggaaatggaa
660ccataatgca catcacattg cctgtaatag cctcgattac gacccggatt tgcagtacat
720tccgtttctt gttgtgtcgt ccaagttgtt tagctcgctc acctctcatt tctatgaaaa
780gaaactgaca tttgactctt tatcgagatt ctttgtaagc catcagcatt ggacgtttta
840cccggttatg tgtatggcta gggttaatat gtttgtgcag tctctgataa tgttgttgac
900taagcgaaat gtgttctata gaagtcaaga actgttggga ttggtggtgt tttggatttg
960gtacccgttg cttgtttctt gcttgcctaa ttggggagaa cgagtaatgt tcgttgttgc
1020tagtctctcg gtgactggaa tgcaacaagt gcagttctct ttgaaccatt tctcgtcgag
1080tgtttatgtt ggtcagccta aagggaacga ttggttcgag aaacaaacat gtgggacgct
1140cgacatttct tgcccttcgt ggatggattg gtttcatggt ggattgcaat tccaagttga
1200gcatcatttg ttccctaagc tgcccagatg ccaccttcgg aaaatctccc cgttcgtgat
1260ggagttatgc aagaagcata atttgtctta caattgtgca tctttctccg aggccaacaa
1320tatgacactc agaacattaa gggacacagc attgcaagct cgcgatttaa ccaagccgct
1380ccccaagaat ttggtatggg aagctcttaa tactcatggt tgagc
142526390PRTPerilla frutescens 26Met Ala Val Ser Ser Gly Ala Arg Leu Ser
Lys Ser Gly Ala Asp Gly 1 5 10
15 Glu Val Phe Asp Gly Gln Gln Gln Tyr Glu Gly Ile Gly Lys Arg
Ala 20 25 30 Ala
Asp Lys Phe Asp Pro Ala Ala Pro Pro Pro Phe Lys Ile Ala Asp 35
40 45 Ile Arg Ala Ala Ile Pro
Ala His Cys Trp Val Lys Ser Pro Trp Arg 50 55
60 Ser Leu Ser Tyr Val Val Trp Asp Val Ala Ala
Val Ser Ala Arg Pro 65 70 75
80 Ala Ala Val Tyr Ile Asn Ser Trp Ala Phe Trp Pro Val Tyr Trp Ile
85 90 95 Ala Gln
Gly Thr Met Phe Trp Ala Leu Phe Val Leu Gly His Asp Cys 100
105 110 Gly His Gly Ser Phe Ser Asp
Asn Thr Thr Leu Asn Asn Val Val Gly 115 120
125 His Val Leu His Ser Ser Ile Leu Val Pro Tyr His
Gly Trp Arg Ile 130 135 140
Ser His Arg Thr His His Gln Asn His Gly His Val Glu Lys Asp Glu 145
150 155 160 Ser Trp Val
Pro Leu Pro Glu Asn Leu Tyr Lys Lys Leu Asp Phe Ser 165
170 175 Thr Lys Phe Leu Arg Tyr Lys Ile
Pro Phe Pro Met Phe Ala Tyr Pro 180 185
190 Leu Tyr Leu Trp Tyr Arg Ser Pro Gly Lys Thr Gly Ser
His Phe Asn 195 200 205
Pro Tyr Ser Asp Leu Phe Lys Pro Asn Glu Arg Gly Leu Ile Val Thr 210
215 220 Ser Thr Met Cys
Trp Ala Ala Met Gly Val Phe Leu His Tyr Ala Thr 225 230
235 240 Thr Ile Val Gly Pro Asn Met Met Phe
Lys Leu Tyr Gly Val Pro Tyr 245 250
255 Leu Ile Phe Val Met Trp Leu Asp Thr Val Thr Tyr Leu His
His His 260 265 270
Gly Tyr Asp Lys Lys Leu Pro Trp Tyr Arg Ser Lys Glu Trp Ile Tyr
275 280 285 Leu Arg Gly Gly
Leu Thr Thr Val Asp Gln Asp Tyr Gly Phe Phe Asn 290
295 300 Lys Ile His His Asp Ile Gly Thr
His Val Ile His His Leu Phe Pro 305 310
315 320 Gln Ile Pro His Tyr His Leu Val Glu Ala Thr Arg
Glu Ala Lys Arg 325 330
335 Val Leu Gly Asn Tyr Tyr Arg Glu Pro Arg Lys Ser Gly Pro Val Pro
340 345 350 Leu His Leu
Ile Pro Ala Leu Leu Lys Ser Leu Gly Arg Asp His Tyr 355
360 365 Val Ser Asp Asn Gly Asp Ile Val
Tyr Tyr His Thr Val Asp Glu Leu 370 375
380 Phe Pro Ser Lys Lys Ile 385 390
27383PRTBrassica napus 27Met Val Val Ala Met Asp Gln Arg Ser Asn Val Asn
Gly Asp Ser Gly 1 5 10
15 Ala Arg Lys Glu Glu Gly Phe Asp Pro Ser Ala Gln Pro Pro Phe Lys
20 25 30 Ile Gly Asp
Ile Arg Ala Ala Ile Pro Lys His Cys Trp Val Lys Ser 35
40 45 Pro Leu Arg Ser Met Ser Tyr Val
Thr Arg Asp Ile Phe Ala Val Ala 50 55
60 Ala Leu Ala Met Ala Ala Val Tyr Phe Asp Ser Trp Phe
Leu Trp Pro 65 70 75
80 Leu Tyr Trp Val Ala Gln Gly Thr Leu Phe Trp Ala Ile Phe Val Leu
85 90 95 Gly His Asp Cys
Gly His Gly Ser Phe Ser Asp Ile Pro Leu Leu Asn 100
105 110 Ser Val Val Gly His Ile Leu His Ser
Phe Ile Leu Val Pro Tyr His 115 120
125 Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn His Gly
His Val 130 135 140
Glu Asn Asp Glu Ser Trp Val Pro Leu Pro Glu Lys Leu Tyr Lys Asn 145
150 155 160 Leu Pro His Ser Thr
Arg Met Leu Arg Tyr Thr Val Pro Leu Pro Met 165
170 175 Leu Ala Tyr Pro Ile Tyr Leu Trp Tyr Arg
Ser Pro Gly Lys Glu Gly 180 185
190 Ser His Phe Asn Pro Tyr Ser Ser Leu Phe Ala Pro Ser Glu Arg
Lys 195 200 205 Leu
Ile Ala Thr Ser Thr Thr Cys Trp Ser Ile Met Leu Ala Thr Leu 210
215 220 Val Tyr Leu Ser Phe Leu
Val Asp Pro Val Thr Val Leu Lys Val Tyr 225 230
235 240 Gly Val Pro Tyr Ile Ile Phe Val Met Trp Leu
Asp Ala Val Thr Tyr 245 250
255 Leu His His His Gly His Asp Glu Lys Leu Pro Trp Tyr Arg Gly Lys
260 265 270 Glu Trp
Ser Tyr Leu Arg Gly Gly Leu Thr Thr Ile Asp Arg Asp Tyr 275
280 285 Gly Ile Phe Asn Asn Ile His
His Asp Ile Gly Thr His Val Ile His 290 295
300 His Leu Phe Pro Gln Ile Pro His Tyr His Leu Val
Asp Ala Thr Arg 305 310 315
320 Ala Ala Lys His Val Leu Gly Arg Tyr Tyr Arg Glu Pro Lys Thr Ser
325 330 335 Gly Ala Ile
Pro Ile His Leu Val Glu Ser Leu Val Ala Ser Ile Lys 340
345 350 Lys Asp His Tyr Val Ser Asp Thr
Gly Asp Ile Val Phe Tyr Glu Thr 355 360
365 Asp Pro Asp Leu Tyr Val Tyr Ala Ser Asp Lys Ser Lys
Ile Asn 370 375 380
28386PRTBetula pendula 28Met Lys Glu Pro Val Leu Glu Glu Met Glu Asn Ala
Gly Gly Phe Gly 1 5 10
15 Asn Gly Phe His Gly Val Val Glu Lys Asp Asp Phe Asp Pro Ser Ala
20 25 30 Pro Pro Pro
Phe Lys Ile Ala Glu Ile Arg Ala Ala Ile Pro Lys His 35
40 45 Cys Trp Ala Lys Asn Pro Trp Arg
Ser Leu Ser Tyr Ala Leu Arg Asp 50 55
60 Val Phe Val Val Ile Ala Leu Ala Ala Ala Ala Ile Tyr
Phe Lys Ala 65 70 75
80 Trp Ile Phe Trp Pro Leu Tyr Trp Ala Ala Gln Gly Thr Met Phe Trp
85 90 95 Ala Leu Phe Val
Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asp 100
105 110 Asn Pro Glu Leu Asn Asn Leu Val Gly
His Val Leu His Ser Ala Ile 115 120
125 Leu Val Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr His
His Gln 130 135 140
Asn His Gly Asn Val Glu Asn Asp Glu Ser Trp Val Pro Leu Thr Glu 145
150 155 160 Lys Leu Tyr Lys Ser
Leu Gly Tyr Ser Thr Arg Leu Leu Arg Phe Thr 165
170 175 Val Pro Phe Pro Leu Phe Ala Tyr Pro Ile
Tyr Leu Trp Ser Arg Ser 180 185
190 Pro Gly Lys Glu Gly Ser His Phe Asn Pro Tyr Ser Asn Leu Phe
Ser 195 200 205 Pro
Asn Glu Arg Lys Asp Val Ile Thr Ser Thr Leu Cys Trp Ser Leu 210
215 220 Met Ala Ala Leu Leu Ile
Tyr Ser Ser Cys Ala Ile Gly Pro Ile Gln 225 230
235 240 Met Leu Lys Leu Tyr Gly Val Pro His Leu Ile
Phe Val Met Trp Leu 245 250
255 Asp Leu Val Thr Tyr Leu His His His Gly Tyr Glu Gln Lys Leu Pro
260 265 270 Trp Tyr
Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr 275
280 285 Val Asp Arg Asp Tyr Gly Trp
Phe Asn Asn Ile His His Asp Ile Gly 290 295
300 Thr His Val Ile His His Leu Phe Pro Gln Ile Pro
His Tyr His Leu 305 310 315
320 Val Glu Ala Thr Asn Ala Ala Lys Pro Val Leu Gly Lys Tyr Tyr Arg
325 330 335 Glu Pro Lys
Arg Ser Gly Pro Phe Pro Ile His Leu Ile Lys Asn Leu 340
345 350 Val Arg Ser Ile Ser Glu Asp His
Tyr Val Asn Asp Asn Gly Asp Ile 355 360
365 Val Tyr Tyr Gln Thr Asp Pro Glu Leu Tyr Lys Ser Ser
Asn Thr Lys 370 375 380
Ser Asp 385 29386PRTArabidopsis thaliana 29Met Val Val Ala Met Asp
Gln Arg Thr Asn Val Asn Gly Asp Pro Gly 1 5
10 15 Ala Gly Asp Arg Lys Lys Glu Glu Arg Phe Asp
Pro Ser Ala Gln Pro 20 25
30 Pro Phe Lys Ile Gly Asp Ile Arg Ala Ala Ile Pro Lys His Cys
Trp 35 40 45 Val
Lys Ser Pro Leu Arg Ser Met Ser Tyr Val Val Arg Asp Ile Ile 50
55 60 Ala Val Ala Ala Leu Ala
Ile Ala Ala Val Tyr Val Asp Ser Trp Phe 65 70
75 80 Leu Trp Pro Leu Tyr Trp Ala Ala Gln Gly Thr
Leu Phe Trp Ala Ile 85 90
95 Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asp Ile Pro
100 105 110 Leu Leu
Asn Ser Val Val Gly His Ile Leu His Ser Phe Ile Leu Val 115
120 125 Pro Tyr His Gly Trp Arg Ile
Ser His Arg Thr His His Gln Asn His 130 135
140 Gly His Val Glu Asn Asp Glu Ser Trp Val Pro Leu
Pro Glu Arg Val 145 150 155
160 Tyr Lys Lys Leu Pro His Ser Thr Arg Met Leu Arg Tyr Thr Val Pro
165 170 175 Leu Pro Met
Leu Ala Tyr Pro Leu Tyr Leu Cys Tyr Arg Ser Pro Gly 180
185 190 Lys Glu Gly Ser His Phe Asn Pro
Tyr Ser Ser Leu Phe Ala Pro Ser 195 200
205 Glu Arg Lys Leu Ile Ala Thr Ser Thr Thr Cys Trp Ser
Ile Met Phe 210 215 220
Val Ser Leu Ile Ala Leu Ser Phe Val Phe Gly Pro Leu Ala Val Leu 225
230 235 240 Lys Val Tyr Gly
Val Pro Tyr Ile Ile Phe Val Met Trp Leu Asp Ala 245
250 255 Val Thr Tyr Leu His His His Gly His
Asp Glu Lys Leu Pro Trp Tyr 260 265
270 Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr
Ile Asp 275 280 285
Arg Asp Tyr Gly Ile Phe Asn Asn Ile His His Asp Ile Gly Thr His 290
295 300 Val Ile His His Leu
Phe Pro Gln Ile Pro His Tyr His Leu Val Asp 305 310
315 320 Ala Thr Lys Ala Ala Lys His Val Leu Gly
Arg Tyr Tyr Arg Glu Pro 325 330
335 Lys Thr Ser Gly Ala Ile Pro Ile His Leu Val Glu Ser Leu Val
Ala 340 345 350 Ser
Ile Lys Lys Asp His Tyr Val Ser Asp Thr Gly Asp Ile Val Phe 355
360 365 Tyr Glu Thr Asp Pro Asp
Leu Tyr Val Tyr Ala Ser Asp Lys Ser Lys 370 375
380 Ile Asn 385
3046DNAArtificialOligonucleotide primer 30atccccgggt accggtcgcc
accatggcta atgcaatcaa gaagta
463131DNAArtificialOligonucleotide primer 31ttggagctca accatgagta
ttaagagctt c 31
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