Patent application title: SUGAR AND LIPID METABOLISM REGULATORS IN PLANTS VI
Oliver Oswald (Ludwigshafen, DE)
JÖrg Bauer (Ludwigshafen, DE)
JÖrg Bauer (Ludwigshafen, DE)
Thorsten Zank (Mannheim, DE)
BASF Plant Science GmbH
IPC8 Class: AA01H500FI
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide alters fat, fatty oil, ester-type wax, or fatty acid production in the plant
Publication date: 2010-02-04
Patent application number: 20100031392
Isolated nucleic acids and proteins associated with lipid and sugar
metabolism regulation are provided. In particular, lipid metabolism
proteins (LMP) and encoding nucleic acids originating from Arabidopsis
thaliana, Brassica napus, and Helianthus annuus are provided. The nucleic
acids and proteins are used in methods of producing transgenic plants and
modulating levels of seed storage compounds. Preferably, the seed storage
compounds are lipids, fatty acids, starches, or seed storage proteins.
1. An isolated lipid metabolism protein (LMP) nucleic acid comprising a
polynucleotide sequence selected from the group consisting of:a) the
polynucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 123,b) a
polynucleotide sequence encoding the polypeptide of SEQ ID NO: 12,c) a
polynucleotide sequence having at least 90% sequence identity to the
polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 123,d) a polynucleotide
sequence encoding a polypeptide having at least 90% sequence identity to
the polypeptide of SEQ ID NO: 12,e) a polynucleotide sequence that
hybridizes under stringent conditions to the full-length polynucleotide
sequence of a) or b) above, wherein the stringent conditions comprise
hybridization in a 6.times. sodium chloride/sodium citrate (6.times.SSC)
solution at 65.degree. C., andf) a polynucleotide sequence that is
complementary to the full-length polynucleotide sequence of a) or b)
above,wherein expression of said polynucleotide in a plant results in an
increase in total fatty acids in seed of said plant.
2. The isolated LMP nucleic acid of claim 1, wherein the polynucleotide sequence is shown in SEQ ID NO: 11 or SEQ ID NO: 123, or the polynucleotide sequence encodes the polypeptide sequence as shown in SEQ ID NO: 12.
3. The isolated LMP nucleic acid of claim 1, wherein the nucleic acid comprises a polynucleotide sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 123, or encoding a polypeptide having at least 90% identity to the polypeptide of SEQ ID NO: 12, wherein expression of said polynucleotide in a plant results in an increase in total fatty acids in seed of said plant.
4. An expression vector comprising the LMP nucleic acid of claim 1.
5. An expression vector comprising an LMP nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of:a) the polynucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 123,b) a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 12,c) a polynucleotide sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 123, andd) a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 12,wherein expression of said polynucleotide in a plant results in an increase in total fatty acids in seed of said plant.
6. The expression vector of claim 4, wherein the LMP nucleic acid is operatively linked to a heterologous promoter selected from the group consisting of a seed-specific promoter, a root-specific promoter, and a non-tissue-specific promoter.
7. A transgenic plant cell comprising the expression vector of claim 4.
8. A transgenic plant comprising the expression vector of claim 4.
9. The transgenic plant of claim 8, wherein the plant is a dicotyledonous plant.
10. The transgenic plant of claim 8, wherein the plant is a monocotyledonous plant.
11. The transgenic plant of claim 8, wherein the plant is selected from the group consisting of rapeseed, canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, sugarbeet, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut.
12. A seed produced by the transgenic plant of claim 8, wherein the plant is true breeding for an increased level of total fatty acids in the seeds of the plant as compared to the seeds of a control plant, and wherein the control plant is of the same ecotype as the transgenic plant but has not been transformed with the LMP nucleic acid, and wherein said seed comprises said expression vector.
13. A method of producing a transgenic plant having an increased level of total fatty acids in the seeds of said plant, said method comprising the steps of transforming a plant cell with the expression vector of claim 4 and generating from the plant cell a transgenic plant having an increased level of total fatty acids in the seeds.
14. The method of claim 13, wherein the increased level of total fatty acids is due to the overexpression of the LMP nucleic acid, wherein said nucleic acid is operatively linked to a promoter selected from the group consisting of constitutive promoters, inducible promoters, tissue-specific promoters, seed-specific promoters, and root-specific promoters.
15. The method of claim 13, wherein the LMP nucleic acid encodes a polypeptide comprising a domain selected from the group consisting of an RNA binding domain, a signal transduction domain, a lipid metabolism domain, an oxidoreductase domain, a fatty acid metabolism domain, a protein synthesis domain, an alkaloid biosynthesis domain, a biosynthesis precursor supply domain, and an intracellular transport domain.
16. The method of claim 13, wherein the polynucleotide sequence is shown in SEQ ID NO: 11 or SEQ ID NO: 123, or the polynucleotide sequence encodes the polypeptide sequence as shown in SEQ ID NO: 12.
17. The method of claim 13, wherein the nucleic acid comprises a polynucleotide sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 123, or encoding a polypeptide having at least 90% identity to the polypeptide of SEQ ID NO: 12, wherein expression of said polynucleotide in a plant results in an increase in total fatty acids in seed of said plant.
18. A method of increasing the level of total fatty acids in the seeds of a plant comprising increasing the expression of an LMP nucleic acid in the plant by transforming the plant with the expression vector of claim 4.
19. The method of claim 18, the polynucleotide sequence is shown in SEQ ID NO: 11 or SEQ ID NO: 123, or the polynucleotide sequence encodes the polypeptide sequence as shown in SEQ ID NO: 12.
20. The method of claim 18, wherein the nucleic acid comprises a polynucleotide sequence having at least 90% sequence identity to the polynucleotide of SEQ ID NO: 11 or SEQ ID NO: 123, or encoding a polypeptide having at least 90% identity to the polypeptide of SEQ ID NO: 12, wherein expression of said polynucleotide in a plant results in an increase in total fatty acids in seed of said plant.
This application is a divisional of application Ser. No. 11/788,342, filed Apr. 19, 2007, which is a divisional of application Ser. No. 11/021,619, filed Dec. 23, 2004, which claims benefit to U.S. Provisional Application Ser. No. 60/532,751, filed Dec. 23, 2003. The entire content of each above-mentioned application is hereby incorporated by reference in entirety.
SUBMISSION OF SEQUENCE LISTING
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing--12810--00926. The size of the text file is 162 KB, and the text file was created on Aug. 27, 2009.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to nucleic acid sequences encoding proteins that are related to the presence of seed storage compounds in plants. More specifically, the present invention relates to nucleic acid sequences encoding sugar, protein, and lipid metabolism enzymes and regulator proteins and the use of these sequences in transgenic plants. The invention further relates to methods of applying these novel plant polypeptides to the identification and stimulation of plant growth and/or to the increase of yield of seed storage compounds.
2. Background Art
The study and genetic manipulation of plants has a long history that began even before the famed studies of Gregor Mendel. In perfecting this science, scientists have accomplished modification of particular traits in plants ranging from potato tubers having increased starch content to oilseed plants such as canola and sunflower having increased or altered fatty acid content. With the increased consumption and use of plant oils, the modification of seed oil content and seed oil levels has become increasingly widespread (e.g. Topfer et al., 1995, Science 268:681-686). Manipulation of biosynthetic pathways in transgenic plants provides a number of opportunities for molecular biologists and plant biochemists to affect plant metabolism giving rise to the production of specific higher-value products. The seed oil production or composition has been altered in numerous traditional oilseed plants such as soybean (U.S. Pat. No. 5,955,650), canola (U.S. Pat. No. 5,955,650), sunflower (U.S. Pat. No. 6,084,164), rapeseed (Topfer et al., 1995, Science 268:681-686), and non-traditional oil seed plants such as tobacco (Cahoon et al., 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).
Plant seed oils comprise both neutral and polar lipids (See Table 1). The neutral lipids contain primarily triacylglycerol, which is the main storage lipid that accumulates in oil bodies in seeds. The polar lipids are mainly found in the various membranes of the seed cells, e.g. the microsomal, plastidial, and mitochondrial membranes, and the cell membrane. The neutral and polar lipids contain several common fatty acids (See Table 2) and a range of less common fatty acids. The fatty acid composition of membrane lipids is highly regulated and only a select number of fatty acids are found in membrane lipids. On the other hand, a large number of unusual fatty acids can be incorporated into the neutral storage lipids in seeds of many plant species (Van de Loo F. J. et al., 1993, Unusual Fatty Acids in Lipid Metabolism in Plants pp. 91-126, editor T S Moore Jr. CRC Press; Millar et al., 2000, Trends Plant Sci. 5:95-101).
TABLE-US-00001 TABLE 1 Plant Lipid Classes Neutral Lipids Triacylglycerol (TAG) Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar Lipids Monogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG) Phosphatidylglycerol (PG) Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylinositol (PI) Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol
TABLE-US-00002 TABLE 2 Common Plant Fatty Acids 16:0 Palmitic acid 16:1 Palmitoleic acid 16:3 Hiragonic acid 18:0 Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 18:3 Linolenic acid γ-18:3.sup. Gamma-linolenic acid * 20:0 Arachidic acid 20:1 Eicosenoic acid 22:6 Docosahexanoic acid (DHA) * 20:2 Eicosadienoic acid 20:4 Arachidonic acid (AA) * 20:5 Eicosapentaenoic acid (EPA) * 22:1 Erucic acid
In Table 2, the fatty acids denoted with an asterisk do not normally occur in plant seed oils, but their production in transgenic plant seed oil is of importance in plant biotechnology.
The primary sites of fatty acid biosynthesis in plants are the plastids. Fatty acid biosynthesis begins with the conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACCase). The malonyl moiety is then transferred to an acyl carrier protein (ACP) by the malonyl-CoA:ACP transacylase. The enzyme beta-keto-acyl-ACP-synthase III (KAS III) catalyzes the initial condensation reaction of fatty acid biosynthesis, in which after decarboxylation of malonyl-ACP, the resulting carbanion is transferred to acetyl-CoA by a nucleophilic attack of the carbonyl-carbon, resulting in the formation of 3-ketobutyryl-ACP. The reaction cycle is completed by a reduction, a dehydration and again a reduction yielding butyric acid. This reaction cycle is repeated (with KAS I or KAS II catalyzing the condensation reaction) until the acyl-group reach a chain length of usually 16 to 18 carbon atoms. These acyl-ACPs can be desaturated by the stearoyl-ACP desaturase, used as substrates for plastidial acyltransferases in the formation of lipids through what has been referred to as the prokaryotic pathway, or exported to the cytosol after cleavage from ACP through the action of thioesterases. In the cytosol they enter the acyl-CoA pool and can be used for the synthesis of lipids through what has been referred to as the eukaryotic pathway in the endoplasmic reticulum.
Lipid synthesis through both the prokaryotic and eukaryotic pathways occurs through the successive acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT) and lysophosphatidic acid acyltransfersas (LPAAT) (Browse et al., 1986, Biochemical J. 235:25-31; Ohlrogge & Browse, 1995, Plant Cell 7:957-970). The resulting phosphatidic acid (PA) is the precursor for other polar membrane lipids such as monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQD) in the plastid and phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in the endoplasmic reticulum. The polar lipids are also the sites of further modification of the acyl-chain such as desaturation, acetylenation, and hydroxylation. In the endoplasmic reticulum, PA is also the intermediate in the biosynthesis of triacylglycerol (TAG), the major component of neutral lipids and hence of seed oil. Furthermore, alternative pathways for the biosynthesis of TAGs can exists (i.e. transacylation through the action of phosphatidylcholine:diacylglycerol acyltransferase) (Voelker, 1996, Genetic Engineering ed.: Setlow 18:111-113; Shanklin & Cahoon, 1998, Annu. Rev. Plant Physiol. Plant Mol. Biol. 49:611-641; Frentzen, 1998, Lipids 100:161-166; Millar et al., 2000, Trends Plant Sci. 5:95-101). The reverse reaction, the breakdown of triacylglycerol to diacylglycerol and fatty acids is catalyzed by lipases. Such a breakdown can be seen toward the end of seed development resulting in a certain reduction in seed oil. (Buchanan et al., 2000).
Storage lipids in seeds are synthesized from carbohydrate-derived precursors. Plants have a complete glycolytic pathway in the cytosol (Plaxton, 1996, Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:185-214), and it has been shown that a complete pathway also exists in the plastids of rapeseeds (Kang & Rawsthorne, 1994, Plant J. 6:795-805). Sucrose is the primary source of carbon and energy, transported from the leaves into the developing seeds. During the storage phase of seeds, sucrose is converted in the cytosol to provide the metabolic precursors glucose-6-phosphate and pyruvate. These are transported into the plastids and converted into acetyl-CoA that serves as the primary precursor for the synthesis of fatty acids. Acetyl-CoA in the plastids is the central precursor for lipid biosynthesis. Acetyl-CoA can be formed in the plastids by different reactions, and the exact contribution of each reaction is still being debated (Ohlrogge & Browse, 1995, Plant Cell 7:957-970). It is accepted, however, that a large part of the acetyl-CoA is derived from glucose-6-phospate and pyruvate that are imported from the cytoplasm into the plastids. Sucrose is produced in the source organs (leaves, or anywhere that photosynthesis occurs) and is transported to the developing seeds that are also termed sink organs. In the developing seeds, the sucrose is the precursor for all the storage compounds, i.e. starch, lipids, and partly the seed storage proteins. Therefore, it is clear that carbohydrate metabolism in which sucrose plays a central role is very important to the accumulation of seed storage compounds.
Although lipid and fatty acid content of seed oil can be modified by the traditional methods of plant breeding, the advent of recombinant DNA technology has allowed for easier manipulation of the seed oil content of a plant, and in some cases, has allowed for the alteration of seed oils in ways that could not be accomplished by breeding alone (See, e.g., Topfer et al., 1995, Science 268:681-686). For example, introduction of a Δ12-hydroxylase nucleic acid sequence into transgenic tobacco resulted in the formation of a novel fatty acid, ricinoleic acid, into the tobacco seed oil (Van de Loo et al., 1995, Proc. Natl. Acad. Sci. USA 92:6743-6747). Tobacco plants have also been engineered to produce low levels of petroselinic acid by the introduction and expression of an acyl-ACP desaturase from coriander (Cahoon et al., 1992, Proc. Natl. Acad. Sci. USA 89:11184-11188).
The modification of seed oil content in plants has significant medical, nutritional, and economic ramifications. With regard to the medical ramifications, the long chain fatty acids (C18 and longer) found in many seed oils have been linked to reductions in hypercholesterolemia and other clinical disorders related to coronary heart disease (Brenner, 1976, Adv. Exp. Med. Biol. 83:85-101). Therefore, consumption of a plant having increased levels of these types of fatty acids may reduce the risk of heart disease. Enhanced levels of seed oil content also increase large-scale production of seed oils and thereby reduce the cost of these oils.
In order to increase or alter the levels of compounds such as seed oils in plants, nucleic acid sequences and proteins regulating lipid and fatty acid metabolism must be identified. As mentioned earlier, several desaturase nucleic acids such as the Δ6-desaturase nucleic acid, Δ12-desaturase nucleic acid and acyl-ACP desaturase nucleic acid have been cloned and demonstrated to encode enzymes required for fatty acid synthesis in various plant species. Oleosin nucleic acid sequences from such different species as Brassica, soybean, carrot, pine, and Arabidopsis thaliana have also been cloned and determined to encode proteins associated with the phospholipid monolayer membrane of oil bodies in those plants.
Although several compounds are known that generally affect plant and seed development, there is a clear need to specifically identify factors that are more specific for the developmental regulation of storage compound accumulation and to identify genes which have the capacity to confer altered or increased oil production to its host plant and to other plant species. This invention discloses nucleic acid sequences from Arabidopsis thaliana, Brassica napus, and Helianthus annuus. These nucleic acid sequences can be used to alter or increase the levels of seed storage compounds such as proteins, sugars, and oils, in plants, including transgenic plants, such as rapeseed, canola, linseed, soybean, sunflower maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor and peanut, which are oilseed plants containing high amounts of lipid compounds.
SUMMARY OF THE INVENTION
The present invention provides novel isolated nucleic acid and amino acid sequences associated with the metabolism of seed storage compounds in plants.
The present invention also provides isolated nucleic acids from Arabidopsis thaliana, Brassica napus, and Helianthus annuus encoding Lipid Metabolism Proteins (LMPs), or portions thereof. These sequences may be used to modify or increase lipids and fatty acids, cofactors, sugars, and enzymes in microorganisms and plants.
Arabidopsis plants are known to produce considerable amounts of fatty acids such as linoleic and linolenic acid (See, e.g., Table 2) and for their close similarity in many aspects (gene homology, etc.) to the oil crop plant Brassica. Therefore, nucleic acid molecules originating from a plant like Arabidopsis thaliana are especially suited to modify the lipid and fatty acid metabolism in a host, especially in microorganisms and plants. Furthermore, nucleic acids from the plants Arabidopsis thaliana can be used to identify those DNA sequences and enzymes in other species which are useful to modify the biosynthesis of precursor molecules of fatty acids in the respective organisms.
The present invention also provides isolated nucleic acids comprising a fragment of at least 60 nucleotides of an Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP nucleic acid disclosed herein. The present invention further provides isolated nucleic acids having at least 70% sequence identity with a full-length Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP nucleic acid disclosed herein. The present invention also provides isolated nucleic acids having at least 90% sequence identity with a full-length Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP nucleic acid disclosed herein. The present invention further provides isolated nucleic acids that hybridize under stringent conditions to an Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP nucleic acid disclosed herein.
Also provided by the present invention are polypeptides encoded by the nucleic acids, heterologous polypeptides comprising polypeptides encoded by the nucleic acids, and antibodies to those polypeptides. The present invention further provides isolated polypeptides having at least 70% sequence identity with a full-length Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP polypeptide disclosed herein. The present invention also provides isolated polypeptides having at least 90% sequence identity with a full-length Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP polypeptide disclosed herein. Accordingly, the present invention provides novel isolated LMP nucleic acids and isolated LMP polypeptides from Arabidopsis thaliana, Brassica napus, or Helianthus annuus, as well as active fragments, analogs, and orthologs thereof.
Additionally, the present invention relates to and provides the use of LMP nucleic acids in the production of transgenic plants having a modified level of a seed storage compound. A method of producing a transgenic plant with a modified level of a seed storage compound includes the steps of transforming a plant cell with an expression vector comprising an LMP nucleic acid, and generating a plant with a modified level of the seed storage compound from the plant cell. In a preferred embodiment, the plant is an oil producing species or oilseed species selected from the group consisting of rapeseed, canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor, and peanut, for example.
According to the present invention, the compositions and methods described herein can be used to increase or decrease the level of an LMP in a transgenic plant comprising increasing or decreasing the expression of the LMP nucleic acid in the plant. Increased or decreased expression of the LMP nucleic acid can be achieved through in vivo mutagenesis of the LMP nucleic acid. The present invention can also be used to increase or decrease the level of a lipid in a seed oil, to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch in a seed or plant.
Also included herein is a seed produced by a transgenic plant transformed by an LMP DNA sequence, wherein the seed contains the LMP DNA sequence and wherein the plant is true breeding for a modified level of a seed storage compound. The present invention additionally includes a seed oil produced by the aforementioned seed.
Further provided by the present invention are vectors comprising the nucleic acids, host cells containing the vectors, and descendent plant materials produced by transforming a plant cell with the nucleic acids and/or vectors.
According to the present invention, the compounds, compositions, and methods described herein can be used to increase or decrease the level of a lipid in a seed oil, or to increase or decrease the level of a fatty acid in a seed oil, or to increase or decrease the level of a starch or other carbohydrate in a seed or plant. A method of producing a higher or lower than normal or typical level of storage compound in a transgenic plant, comprises expressing an LMP nucleic acid from Arabidopsis thaliana, Brassica napus, or Helianthus annuus in the transgenic plant, wherein the transgenic plant is Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or a species different from Arabidopsis thaliana, Brassica napus, or Helianthus annuus. Also included herein are compositions and methods of the modification of the efficiency of production of a seed storage compound.
The present invention also provides transgenic plants having modified levels of seed storage compounds, and in particular, modified levels of a protein, a lipid, a fatty acid, or a sugar.
The polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof also have uses that include modulating plant growth, and potentially plant yield, preferably increasing plant growth under adverse conditions (drought, cold, light, UV). In addition, antagonists of the present invention may have uses that include modulating plant growth and/or yield, preferably through increasing plant growth and yield. In yet another embodiment, overexpression of the polypeptides of the present invention using a constitutive promoter (e.g., 35S or other promoters) may be useful for increasing plant yield under stress conditions (drought, light, cold, UV) by modulating light utilization efficiency.
The present invention also provides methods for producing such aforementioned transgenic plants. The present invention further provides seeds and seed oils from such aforementioned transgenic plants.
These and other embodiments, features, and advantages of the present invention will become apparent after a review of the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the binary vector carrying the osw20 suppression construct.
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein.
Before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, provides an" isolated nucleic acid from a plant (Arabidopsis thaliana, Brassica napus, and Helianthus annuus) encoding a Lipid Metabolism Protein (LMP), or a portion thereof. As used herein, the phrase "Arabidopsis thaliana, Brassica napus, and Helianthus annuus" is intended to mean Arabidopsis thaliana and/or Brassica napus and/or Helianthus annuus.
One aspect of the invention pertains to isolated nucleic acid molecules that encode LMP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of an LMP-encoding nucleic acid (e.g., LMP DNA). As used herein, the terms "nucleic acid molecule" and "polynucleotide sequence" are used interchangeably and are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3' and 5' ends of the coding region of a gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An "isolated" nucleic acid molecule is one which is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is substantially free of sequences, which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated LMP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., an Arabidopsis thaliana, Brassica napus, or Helianthus annuus cell). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors, or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a polynucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, an Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP cDNA can be isolated from an Arabidopsis thaliana, Brassica napus, or Helianthus annuus library using all or portion of one of the disclosed polynucleotide sequences as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Moreover, a nucleic acid molecule encompassing all or a portion of one of the disclosed sequences can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence. For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al., 1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the polynucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an LMP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid of the invention comprises one of the polynucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123. These polynucleotides correspond to the Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP cDNAs of the invention. These cDNAs comprise sequences encoding LMPs (i.e., the "coding region" or open reading frame (ORF)). Alternatively, the nucleic acid molecules also may comprise 5' untranslated sequences and 3' untranslated sequences of the polynucleotide sequences described herein or can contain whole genomic fragments isolated from genomic DNA. The particular polynucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123 have been given an identifying entry number (e.g. Osw14). These sequences represent the coding region or open reading frame, and the putative functions of the encoded polypeptides are indicated in Table 3.
TABLE-US-00003 TABLE 3 Putative LMP Functions Sequence ORF code Function position osw14 RNA binding factor 1-2976 osw15 Regulator of proteasomes and transcription factors 1-1014 osw16 γ-COP/Adaptin 1-2661 osw17 Strictosidine synthase 1-1131 osw18 Strictosidine synthase 1-1113 osw20 GDSL-like lipase 1-1119 osw21 Retinaldehyde binding prot. 1-447 osw22 Lipid binding protein 1-1134 osw23 Lipocalin and fattay acid binding protein 1-594 osw24 Diacylglycerol binding protein 1-780 osw26 Beta-hydroxysteroid dehydrogenase 1-915 JB69 Aspartate protease 1-1521 JB70 Aspartate protease 1-1542 JB71 Aspartate protease 1-1527 JB080 Palmitoyl-protein thioesterase precursor-like 1-951 JB082 Laccase (diphenol oxidase) 1-1473 JB084 Alanine aminotransferase, putative 1-1470 JB085 Peptidylprolyl isomerase 1-519 JB088 Cysteine proteinase-like protein 1-1095 JB089 Plant invertase/pectin methylesterase inhibitor 1-516 JB090 Gamma interferon inducible lysosomal thiol 1-702 reductase JB091 Blue copper binding-like protein 1-591 JB093 Embryonic abundant protein 1-459 ToZ001 Glycerinaldehyde-3-phosphate dehydrogenase 1-1191 ToZ002 Glycerol kinase 1-1569 ToZ003 Phosphomannomutase 1-741 ToZ004 Choline/ethanolamine phosphotransferase 1-1170 ToZ005 Choline/ethanolamine kinase 1-2019 ToZ011 Fatty acid elongase ELO-like 1-897 ToZ012 Fatty acid elongase ELO-like 1-837
TABLE-US-00004 TABLE 4 Grouping of LMPs Based on Functional Protein Domains Domain Functional SEQ SEQ position category ID: Code: Functional domain (aa) Fatty acid 29 JB80 Palmitoyl protein thioesterase (PFAM) 21-297 metabolism 57 ToZ11 GNS1/SUR4 family (PFAM) 1-277 59 ToZ12 GNS1/SUR4 family (PFAM) 1-277 Intracellular 5 Osw16 Adaptin N (PFAM) 25-527 transport 51 ToZ03 Eukaryotic phosphomannomutase (PFAM) 29-246 11-234 Lipid 11 Osw20 GDSL-like Lipase/Acylhydrolase (PFAM) 37-333 metabolism 13 Osw21 (acyl-carrier-protein) S-malonyltransferase (COG) 3-147 Cellular retinaldehyde-binding protein (BLOCKS) 111-118 15 Osw22 Arabidopsis protein of unknown function (PFAM) 125-236 Cellular retinaldehyde-binding protein (BLOCKS) 15-29 Sterol regulatory element binding protein site 165-177 (BLOCKS) 19 Osw24 Phorbol esters/diacylglycerol binding domain 22-34 (BLOCKS) 21 Osw26 3-Beta hydroxysteroid dehydrogenase (BLOCKS) 65-95 53 ToZ04 CDP-alcohol phosphatidyltransferase (PFAM) 86-224 55 ToZ05 Choline/ethanolamine kinase (PFAM) 397-655 Oxidoreductases 41 JB90 Gamma interferon inducible lysosomal thiol 32-136 reductase (PFAM) 31 JB82 Multicopper oxidase (PFAM) 72-230 309-472 43 JB91 Type-1 copper (blue) domain (BLOCKS) 105-123 Plastocyanin-like domain (PFAM) 33-117 Precuror 33 JB84 Aminotransferase class I and II (PFAM) 140-480 supply 39 JB89 Plant invertase/pectin methylesterase inhibitor 19-167 (PFAM) 47 ToZ01 Glyceraldehyde 3-phosphate dehydrogenase 61-213 (PFAM) 213-374 49 ToZ02 FGGY family of carbohydrate kinases (PFAM) 7-262 265-501 Proteases 23 JB69 Eukaryotic aspartyl protease (PFAM) 30-505 25 JB70 Eukaryotic aspartyl protease (PFAM) 44-521 27 JB71 Eukaryotic aspartyl protease (PFAM) 41-516 37 JB88 Papain family cysteine protease (PFAM) 137-352 Protein 45 JB93 Small hydrophilic plant seed protein (PFAM) 1-90 stability 92-152 Protein 35 JB85 Cyclophilin type peptidyl-prolyl cis-trans isomerase 5-172 synthesis (PFAM) RNA-binding 1 Osw14 TUDOR (PFAM) 733-806 proteins RNA-binding 17 Osw23 Pumilio-family RNA binding repeat (PFAM) 4-153 proteins/Lipid Lipocalin and cytosolic fatty-acid binding protein 127-137 signal transduction (Blocks) Signal 3 Osw15 Mov34 (PFAM) 20-128 Transduction Alkaloid 7 Osw17 Strictosidine synthase (PFAM) 10-365 biosynthesis 9 Osw18 Strictosidine synthase (PFAM) 5-364
In another preferred embodiment, an isolated nucleic acid molecule of the present invention encodes a polypeptide that is able to participate in the metabolism of seed storage compounds such as lipids, starch, and seed storage proteins, and/or that contains a DNA-binding (or transcription factor) domain, or an RNA-binding domain. Examples of isolated nucleic acids that encode LMPs containing such domains can be found in Table 4. Examples of nucleic acids encoding an LMP with an RNA-binding domain are SEQ ID NO:1 and SEQ ID NO:17. An example of a nucleic acid encoding an LMP with a signal transduction domain includes SEQ ID NO:3. Examples of nucleic acids encoding LMPs containing a protease domain include those shown in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:37. Examples of nucleic acids encoding LMPs containing a lipid metabolism domain include those shown in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:121. Examples of nucleic acids encoding LMPs containing a oxidoreductase domain include those shown in SEQ ID NO:31, SEQ ID NO:41, and SEQ ID NO:43. Examples of nucleic acids encoding LMPs containing a fatty acid metabolism domain include those shown in SEQ ID NO:29, SEQ ID NO:57, and SEQ ID NO:59. Examples of nucleic acids encoding LMPs containing a protein synthesis domain include those shown in SEQ ID NO:35 and SEQ ID NO:45. Examples of nucleic acids encoding LMPs containing an alkaloid biosynthesis domain include those shown in SEQ ID NO:7 and SEQ ID NO:9. Examples of nucleic acids encoding LMPs containing a biosynthesis precursor supply domain include those shown in SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:47, and SEQ ID NO:49. Examples of nucleic acids encoding LMPs containing an intracellular transport domain include those shown in SEQ ID NO:5 and SEQ ID NO:51.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule, which is a complement of one of the polynucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, or a portion thereof. A nucleic acid molecule which is complementary to one of the disclosed polynucleotide sequences is one which is sufficiently complementary to one of the disclosed polynucleotide sequences such that it can hybridize to one of the disclosed polynucleotide sequences, thereby forming a stable duplex.
In another preferred embodiment, an isolated nucleic acid of the invention comprises a polynucleotide sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:122.
In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a polynucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, or more homologous to a full-length polynucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, or a portion thereof. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a polynucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of these disclosed polynucleotide sequences, or a portion thereof. These stringent conditions include washing with a solution having a salt concentration of about 0.02 M at pH 7 and about 60° C. In another embodiment, the stringent conditions comprise hybridization in a 6× sodium chloride/sodium citrate (6×SSC) solution at 45° C. In yet another embodiment, the stringent conditions comprise hybridization in a 6× sodium chloride/sodium citrate (6×SSC) solution at 65° C.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of one of the disclosed sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a LMP. The polynucleotide sequences determined from the cloning of the LMP genes from Arabidopsis thaliana, Brassica napus, and Helianthus annuus allows for the generation of probes and primers designed for use in identifying and/or cloning LMP homologues in other cell types and organisms, as well as LMP homologues from other plants or related species. Therefore this invention also provides compounds comprising the nucleic acids disclosed herein, or fragments thereof. These compounds include the nucleic acids attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50, or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on one of these polynucleotide sequences can be used in PCR reactions to clone LMP homologues. Probes based on the LMP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a LMP, such as by measuring a level of a LMP-encoding nucleic acid in a sample of cells, e.g., detecting LMP mRNA levels or determining whether a genomic LMP gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence encoded by one of the disclosed polynucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:121 such that the protein or portion thereof maintains the same or a similar function as the wild-type protein. As used herein, the language "sufficiently homologous" refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent amino acid residues to an amino acid sequence such that the protein or portion thereof is able to participate in the metabolism of compounds necessary for the production of seed storage compounds in plants, construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes. As used herein, an "equivalent" amino acid residue is, for example, an amino acid residue which has a similar side chain as a particular amino acid residue that is encoded by one of these disclosed polynucleotide sequences. Regulatory proteins, such as RNA binding proteins, protein stability and breakdown proteins, signal transduction proteins, or protein members of metabolic pathways such as the lipid, starch, and protein biosynthetic pathways as well as pathways for the precursor supply of these pathways, or membrane transport systems, may play a role in the biosynthesis of seed storage compounds. Examples of such activities are described herein (see putative annotations in Table 3). Examples of LMP-encoding nucleic acid sequences are SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123.
As altered or increased sugar and/or fatty acid production is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower and tagetes, solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), perennial grasses, and forage crops, these crop plants are also preferred target plants for genetic engineering as one further embodiment of the present invention. As used herein, the term "forage crop" includes, but is not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover. In a preferred embodiment, the plant is an oil producing species or oilseed species selected from the group consisting of rapeseed, canola, linseed, soybean, sunflower, maize, oat, rye, barley, wheat, pepper, tagetes, cotton, oil palm, coconut palm, flax, castor, and peanut, for example. See, e.g., Kinney et al. (1994, Current Opin. in Biotech. 5:144-151), Topfer et al. (1995, Science 268.681-686), and Oil Crops of the World-Their Breeding and Utilization (1989, eds. Robbelen, Downey, and Ashri).
Portions of proteins encoded by the LMP nucleic acid molecules of the invention are preferably biologically active portions of one of the LMPs. As used herein, the term "biologically active portion of an LMP" is intended to include a portion, e.g., a domain/motif, of an LMP that participates in the metabolism of compounds necessary for the biosynthesis of seed storage lipids, or the construction of cellular membranes in microorganisms or plants, or in the transport of molecules across these membranes, or has an activity as set forth in Table 3. To determine whether an LMP or a biologically active portion thereof can participate in the metabolism of compounds necessary for the production of seed storage compounds and cellular membranes, an assay of enzymatic activity may be performed. Such assay methods are well known to those skilled in the art, and specific references are cited in Example 15 herein.
Biologically active portions of an LMP include peptides comprising amino acid sequences derived from the amino acid sequence of an LMP (e.g., an amino acid sequence encoded by a nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123) or the amino acid sequence of a protein homologous to an LMP, which include fewer amino acids than a full length LMP or the full length protein which is homologous to an LMP and exhibits at least one activity of an LMP. Typically, biologically active portions (e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids in length) comprise a domain or motif with at least one activity of an LMP. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an LMP include one or more selected domains/motifs or portions thereof having biological activity.
Additional nucleic acid fragments encoding biologically active portions of an LMP can be prepared by isolating a portion of one of the sequences, expressing the encoded portion of the LMP or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the LMP or peptide.
The invention further encompasses nucleic acid molecules that differ from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123, and portions thereof, due to degeneracy of the genetic code and thus encode the same LMP as that encoded by the disclosed polynucleotide sequences. In a further embodiment, the nucleic acid molecule of the invention encodes a full length polypeptide which is substantially homologous to an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:122. In one embodiment, the full-length nucleic acid or protein or fragment of the nucleic acid or protein is from Arabidopsis thaliana, Brassica napus, and Helianthus annuus.
In addition to the Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP polynucleotide sequences described herein, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of LMPs may exist within a population (e.g., the Arabidopsis thaliana, Brassica napus, and Helianthus annuus population). Such genetic polymorphism in the LMP gene may exist among individuals within a population due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an LMP, preferably an Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP. Such natural variations can typically result in 1-40% variance in the nucleotide sequence of the LMP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in LMP that are the result of natural variation and that do not alter the functional activity of LMPs are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants and non-Arabidopsis thaliana, non-Brassica napus, or non-Helianthus annuus orthologs of the Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP cDNA of the invention can be isolated based on their homology to the Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP nucleic acids disclosed herein using the Arabidopsis thaliana, Brassica napus, or Helianthus annuus cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. As used herein, the term "orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode proteins having the same or similar functions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising one of the polynucleotide sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123. In other embodiments, the nucleic acid is at least 30, 50, 60, 100, 250, or more nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75%, or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. In another embodiment, the stringent conditions comprise hybridization in a 6× sodium chloride/sodium citrate (6×SSC) solution at 45° C. In yet another embodiment, the stringent conditions comprise hybridization in a 6×SSC solution at 65° C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to one of the disclosed polynucleotide sequences corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a polynucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP.
In addition to naturally-occurring variants of the LMP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into one of the disclosed polynucleotide sequences, thereby leading to changes in the amino acid sequence of the encoded LMP, without altering the functional ability of the LMP. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in one of the disclosed polynucleotide sequences. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the disclosed LMPs (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:122) without altering the activity of said LMP, whereas an "essential" amino acid residue is required for LMP activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having LMP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering LMP activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding LMPs that contain changes in amino acid residues that are not essential for LMP activity. Such LMPs differ in amino acid sequence from a sequence yet retain at least one of the LMP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence encoded by a nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123 and is capable of participation in the metabolism of compounds necessary for the production of seed storage compounds in Arabidopsis thaliana, Brassica napus, and Helianthus annuus, or cellular membranes, or has one or more activities set forth in Table 3. Preferably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the sequences encoded by one of the disclosed nucleic acids, more preferably at least about 60-70% homologous to one of the sequences encoded by one of the disclosed nucleic acids, even more preferably at least about 70-80%, 80-90%, or 90-95% homologous to one of the sequences encoded by one of the disclosed nucleic acids, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the sequences encoded by one of the disclosed nucleic acids.
To determine the percent homology of two amino acid sequences (e.g., one of the sequences encoded by a nucleic acid disclosed herein and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the sequences encoded by the disclosed nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the sequence encoded by the disclosed nucleic acid), then the molecules are homologous at that position. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity." The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=numbers of identical positions/total numbers of positions×100).
An isolated nucleic acid molecule encoding an LMP homologous to a protein sequence encoded by a nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:121, or SEQ ID NO:123 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the disclosed polynucleotide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced into one of the disclosed polynucleotide sequences by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in an LMP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an LMP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an LMP activity described herein to identify mutants that retain LMP activity. Following mutagenesis of one of the disclosed polynucleotide sequences, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (See, e.g., Examples 15-16 below).
LMPs are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described herein), the expression vector is introduced into a host cell (as described herein), and the LMP is expressed in the host cell. The LMP can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, an LMP or peptide thereof can be synthesized chemically using standard peptide synthesis techniques. Moreover, native LMP can be isolated from cells, for example, using an anti-LMP antibody which can be produced by standard techniques utilizing an LMP or fragment thereof of this invention.
The invention also provides LMP chimeric or fusion proteins. As used herein, an LMP "chimeric protein" or "fusion protein" comprises an LMP polypeptide operatively linked to a non-LMP polypeptide. An "LMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an LMP, whereas a "non-LMP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the LMP, e.g., a protein which is different from the LMP and which is derived from the same or a different organism. As used herein with respect to the fusion protein, the term "operatively linked" is intended to indicate that the LMP polypeptide and the non-LMP polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-LMP polypeptide can be fused to the N-terminus or C-terminus of the LMP polypeptide. For example, in one embodiment, the fusion protein is a GST-LMP (glutathione S-transferase) fusion protein in which the LMP sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant LMPs. In another embodiment, the fusion protein is an LMP containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an LMP can be increased through use of a heterologous signal sequence.
Preferably, an LMP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (See, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An LMP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the LMP.
In addition to the nucleic acid molecules encoding LMPs described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire LMP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an LMP. The term "coding region" refers to the region of the nucleotide sequence comprising codons, which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding LMP. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding LMP disclosed herein (e.g., the full-length polynucleotide sequences set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:121), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of the LMP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of the LMP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the LMP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense or sense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylamino-methyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydro-uracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl-cytosine, N-6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyl-uracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diamino-purine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
In another variation of the antisense technology, a double-strand interfering RNA construct can be used to cause a down-regulation of the LMP mRNA level and LMP activity in transgenic plants. This requires transforming the plants with a chimeric construct containing a portion of the LMP sequence in the sense orientation fused to the antisense sequence of the same portion of the LMP sequence. A DNA linker region of variable length can be used to separate the sense and antisense fragments of LMP sequences in the construct.
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an LMP to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic including plant promoters are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an anomeric nucleic acid molecule. An anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methyl-ribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff & Gerlach, 1988, Nature 334:585-591)) can be used to catalytically cleave LMP mRNA transcripts to thereby inhibit translation of LMP mRNA. A ribozyme having specificity for an LMP-encoding nucleic acid can be designed based upon the nucleotide sequence of an LMP cDNA disclosed herein or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an LMP-encoding mRNA (See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al.). Alternatively, LMP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (See, e.g., Bartel, D. & Szostak J. W. 1993, Science 261:1411-1418).
Alternatively, LMP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an LMP nucleotide sequence (e.g., an LMP promoter and/or enhancers) to form triple helical structures that prevent transcription of an LMP gene in target cells (See generally, Helene C., 1991, Anticancer Drug Des. 6:569-84; Helene C. et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J., 1992, Bioassays 14:807-15).
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an LMP (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. As used herein with respect to a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence and both sequences are fused to each other so that each fulfills its proposed function (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., eds.: Glick & Thompson, Chapter 7, 89-108 including the references therein. Regulatory sequences include those, which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., LMPs, mutant forms of LMPs, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of LMPs in prokaryotic or eukaryotic cells. For example, LMP genes can be expressed in bacterial cells, insect cells (using baculovirus expression vectors), yeast and other fungal cells (See Romanos M. A. et al., 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C.A.M.J.J. et al., 1991, Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, Bennet & Lasure, eds., p. 396-428:Academic Press: an Diego; and van den Hondel & Punt, 1991, Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatore et al., 1999, Marine Biotechnology 1:239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a transformation method as described in WO 98101572, and multicellular plant cells (See Schmidt & Willmitzer, 1988, High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon plants, Plant Cell Rep.: 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119 (1993); White, Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and Wu, Academic Press 1993, 128-43; Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225 (and references cited therein)), or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve one or more of the following purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith & Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the LMP is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant LMP unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman S., 1990, Gene Expression Technology: Methods in Enzymology 185:119-128, Academic Press, San Diego, Calif.). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression (Wada et al., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the LMP expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., 1987, Embo J. 6:229-234), pMFa (Kurjan & Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel & Punt, 1991, "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy et al., eds., p. 1-28, Cambridge University Press: Cambridge.
Alternatively, the LMPs of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow & Summers, 1989, Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook, Fritsh and Maniatis, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the LMPs of the invention may be expressed in uni-cellular plant cells (such as algae, see Falciatore et al., 1999, Marine Biotechnology 1:239-251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, Kemper, Schell and Masterson (1992, Plant Mol. Biol. 20:1195-1197) and Bevan (1984, "Binary Agrobacterium vectors for plant transformation, Nucleic Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38).
A plant expression cassette preferably contains regulatory sequences capable to drive gene expression in plant cells and which are operatively linked so that each sequence can fulfill its function such as termination of transcription, including polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al. 1984, EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable.
As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al. 1987, Nucleic Acids Res. 15:8693-8711).
Plant gene expression has to be operatively linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Preferred are promoters driving constitutive expression (Benfey et al. 1989, EMBO J. 8:2195-2202) like those derived from plant viruses like the 35S CAMV (Franck et al. 1980, Cell 21:285-294), the 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), or plant promoters like those from Rubisco small subunit described in U.S. Pat. No. 4,962,028. Even more preferred are seed-specific promoters driving expression of LMP proteins during all or selected stages of seed development. Seed-specific plant promoters are known to those of ordinary skill in the art and are identified and characterized using seed-specific mRNA libraries and expression profiling techniques. Seed-specific promoters include, but are not limited to, the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al. 1991, Mol. Gen. Genetics 225:459-67), the oleosin-promoter from Arabidopsis (WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (WO9113980) or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant J. 2:233-239) as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note are the lpt2 or lpt1-gene promoter from barley (WO 95/15389 and WO 95/23230) or those 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, wheat glutelin gene, the maize zein gene, the oat glutelin gene, the Sorghum kasirin-gene, and the rye secalin gene).
Plant gene expression can also be facilitated via an inducible promoter (for review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is desired in a time specific manner. Examples for such promoters are a salicylic acid inducible promoter (WO 95/19443), a tetracycline inducible promoter (Gatz et al. 1992, Plant J. 2:397-404), and an ethanol inducible promoter (WO 93/21334).
Promoters responding to biotic or abiotic stress conditions are also suitable promoters such as the pathogen inducible PRP1-gene promoter (Ward et al., 1993, Plant. Mol. Biol. 22:361-366), the heat inducible hsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold inducible alpha-amylase promoter from potato (WO 96/12814), and the wound-inducible pinII-promoter (EP 375091).
Other preferred sequences for use in plant gene expression cassettes are targeting-sequences necessary to direct the gene-product in its appropriate cell compartment (for review, see Kermode 1996, Crit. Rev. Plant Sci. 15:285-423 and references cited therein) such as the vacuole, the nucleus, all types of plastids like amyloplasts, chloroplasts, chromoplasts, the extracellular space, mitochondria, the endoplasmic reticulum, oil bodies, peroxisomes, and other compartments of plant cells. Also especially suited are promoters that confer plastid-specific gene expression, as plastids are the compartment where precursors and some end products of lipid biosynthesis are synthesized. Suitable promoters such as the viral RNA-polymerase promoter are described in WO 95/16783 and WO 97/06250 and the clpP-promoter from Arabidopsis described in WO 99/46394.
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to LMP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al. (986, Antisense RNA as a molecular tool for genetic analysis, Reviews--Trends in Genetics, Vol. 1) and Mol et al. (1990, FEBS Lett. 268:427-430).
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is to be understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, an LMP can be expressed in bacterial cells, insect cells, fungal cells, mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates or plant cells. Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection," "conjugation," and "transduction" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and other laboratory manuals such as Methods in Molecular Biology 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, N.J.
For stable transfection of mammalian and plant cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, kanamycin, and methotrexate, or in plants that confer resistance towards an herbicide such as glyphosate or glufosinate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an LMP or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an LMP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the LMP gene. Preferably, this LMP gene is an Arabidopsis thaliana, Brassica napus, and Helianthus annuus LMP gene, but it can be a homolog from a related plant or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous LMP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a knock-out vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous LMP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous LMP). To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Res. 27:1323-1330 and Kmiec 1999, American Scientist 87:240-247). Homologous recombination procedures in Arabidopsis thaliana are also well known in the art and are contemplated for use herein.
In a homologous recombination vector, the altered portion of the LMP gene is flanked at its 5' and 3' ends by additional nucleic acid of the LMP gene to allow for homologous recombination to occur between the exogenous LMP gene carried by the vector and an endogenous LMP gene in a microorganism or plant. The additional flanking LMP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas & Capecchi, 1987, Cell 51:503, for a description of homologous recombination vectors). The vector is introduced into a microorganism or plant cell (e.g., via polyethyleneglycol mediated DNA). Cells in which the introduced LMP gene has homologously recombined with the endogenous LMP gene are selected using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of an LMP gene on a vector placing it under control of the lac operon permits expression of the LMP gene only in the presence of IPTG. Such regulatory systems are well known in the art.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture can be used to produce (i.e., express) an LMP. Accordingly, the invention further provides methods for producing LMPs using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (into which a recombinant expression vector encoding an LMP has been introduced, or which contains a wild-type or altered LMP gene in its genome) in a suitable medium until the LMP is produced. In another embodiment, the method further comprises isolating LMPs from the medium or the host cell.
Another aspect of the invention pertains to isolated LMPs, and biologically active portions thereof. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of LMP in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of LMP having less than about 30% (by dry weight) of non-LMP (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-LMP, still more preferably less than about 10% of non-LMP, and most preferably less than about 5% non-LMP. When the LMP or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of LMP in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of LMP having less than about 30% (by dry weight) of chemical precursors or non-LMP chemicals, more preferably less than about 20% chemical precursors or non-LMP chemicals, still more preferably less than about 10% chemical precursors or non-LMP chemicals, and most preferably less than about 5% chemical precursors or non-LMP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the LMP is derived. Typically, such proteins are produced by recombinant expression of, for example, an Arabidopsis thaliana, Brassica napus, or Helianthus annuus LMP in plants other than Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or microorganisms, algae, or fungi.
An isolated LMP or a portion thereof of the invention can participate in the metabolism of compounds necessary for the production of seed storage compounds in Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or of cellular membranes, or has one or more of the activities set forth in Table 3. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence encoded by one of the disclosed nucleic acids shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:121, such that the protein or portion thereof maintains the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or in the transport of molecules across these membranes. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, an LMP of the invention has an amino acid sequence encoded by one of these disclosed nucleic acids. In yet another preferred embodiment, the LMP has an amino acid sequence which is encoded by a polynucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of these disclosed nucleic acids. In still another preferred embodiment, the LMP has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, and even more preferably at least about 96%, 97%, 98%, 99% or more homologous to an amino acid sequence encoded by one of these disclosed nucleic acids. The preferred LMPs of the present invention also preferably possess at least one of the LMP activities described herein. For example, a preferred LMP of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the disclosed nucleic acids, and which can participate in the metabolism of compounds necessary for the construction of cellular membranes in Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or in the transport of molecules across these membranes, or which has one or more of the activities set forth in Table 3.
In other embodiments, the LMP is substantially homologous to an amino acid sequence encoded by a nucleic acid shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:121 and retains the functional activity of the protein encoded by that sequence yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail above. Accordingly, in another embodiment, the LMP is a protein which comprises an amino acid sequence which is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence and which has at least one of the LMP activities described herein. In another embodiment, the invention pertains to a full-length Arabidopsis thaliana, Brassica napus, or Helianthus annuus protein which is substantially homologous to an entire amino acid sequence encoded by a nucleic acid shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:121.
Dominant negative mutations or trans-dominant suppression can be used to reduce the activity of an LMP in transgenics seeds in order to change the levels of seed storage compounds. To achieve this, a mutation that abolishes the activity of the LMP is created and the inactive non-functional LMP gene is overexpressed in the transgenic plant. The inactive trans-dominant LMP protein competes with the active endogenous LMP protein for substrate or interactions with other proteins and dilutes out the activity of the active LMP. In this way the biological activity of the LMP is reduced without actually modifying the expression of the endogenous LMP gene. This strategy was used by Pontier et al. to modulate the activity of plant transcription factors (Pontier et al., 2001, Plant J. 27(6):529-38).
Homologs of the LMP can be generated by mutagenesis, e.g., discrete point mutation or truncation of the LMP. As used herein, the term "homolog" refers to a variant form of the LMP which acts as an agonist or antagonist of the activity of the LMP. An agonist of the LMP can retain substantially the same, or a subset, of the biological activities of the LMP. An antagonist of the LMP can inhibit one or more of the activities of the naturally occurring form of the LMP by, for example, competitively binding to a downstream or upstream member of the cell membrane component metabolic cascade which includes the LMP, or by binding to an LMP which mediates transport of compounds across such membranes, thereby preventing translocation from taking place.
In an alternative embodiment, homologs of the LMP can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the LMP for LMP agonist or antagonist activity. In one embodiment, a variegated library of LMP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of LMP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential LMP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of LMP sequences therein. There are a variety of methods which can be used to produce libraries of potential LMP homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential LMP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983, Nucleic Acids Res. 11:477).
In addition, libraries of fragments of the LMP coding sequences can be used to generate a variegated population of LMP fragments for screening and subsequent selection of homologs of an LMP. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an LMP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal, and internal fragments of various sizes of the LMP.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of LMP homologs. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify LMP homologs (Arkin & Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering 6:327-331).
In another embodiment, cell based assays can be exploited to analyze a variegated LMP library, using methods well known in the art.
The nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Arabidopsis thaliana, Brassica napus, Helianthus annuus, and related organisms; mapping of genomes of organisms related to Arabidopsis thaliana, Brassica napus, or Helianthus annuus; identification and localization of Arabidopsis thaliana, Brassica napus, or Helianthus annuus sequences of interest; evolutionary studies; determination of LMP regions required for function; modulation of an LMP activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; and modulation of seed storage compound accumulation.
The plant Arabidopsis thaliana represents one member of higher (or seed) plants. It is related to other plants such as Brassica napus, Helianthus annuus, and soybean, which require light to drive photosynthesis and growth. Plants like Arabidopsis thaliana and Brassica napus share a high degree of homology on the DNA sequence and polypeptide level, allowing the use of heterologous screening of DNA molecules with probes evolving from other plants or organisms, thus enabling the derivation of a consensus sequence suitable for heterologous screening or functional annotation and prediction of gene functions in third species. The ability to identify such functions can therefore have significant relevance, e.g., prediction of substrate specificity of enzymes. Further, these nucleic acid molecules may serve as reference points for the mapping of Arabidopsis genomes, or of genomes of related organisms.
The LMP nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Arabidopsis thaliana, Brassica napus, or Helianthus annuus, or a close relative thereof. Also, they may be used to identify the presence of Arabidopsis thaliana, Brassica napus, Helianthus annuus, or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of Arabidopsis thaliana, Brassica napus, and Helianthus annuus genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of an Arabidopsis thaliana, Brassica napus, or Helianthus annuus gene which is unique to this organism, one can ascertain whether this organism is present.
Further, the nucleic acid and protein molecules of the invention may serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of Arabidopsis thaliana, Brassica napus, and Helianthus annuus proteins. For example, to identify the region of the genome to which a particular Arabidopsis thaliana, Brassica napus, or Helianthus annuus DNA-binding protein binds, the Arabidopsis thaliana, Brassica napus, or Helianthus annuus genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of Arabidopsis thaliana, Brassica napus, or Helianthus annuus, and when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related plants. Similarly, binding to mRNA sequences by Arabidopsis thaliana, Brassica napus and Helianthus annuus RNA binding proteins of the invention can be determined. In another example of functional studies Arabidopsis thaliana, Brassica napus, and Helianthus annuus proteins of the invention involved in proteolysis and protein stability can be used to identify partners in protein-protein interaction assays, for example, in blue native gels or yeast two hybrid screens.
The LMP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.
Manipulation of the LMP nucleic acid molecules of the invention may result in the production of LMPs having functional differences from the wild-type LMPs. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
There are a number of mechanisms by which the alteration of an LMP of the invention may directly affect the accumulation of seed storage compounds. In the case of plants expressing LMPs, increased transport can lead to altered accumulation of compounds and/or solute partitioning within the plant tissue and organs which ultimately could be used to affect the accumulation of one or more seed storage compounds during seed development. An example is provided by Mitsukawa et al. (1997, Proc. Natl. Acad. Sci. USA 94:7098-7102), where overexpression of an Arabidopsis high-affinity phosphate transporter gene in tobacco cultured cells enhanced cell growth under phosphate-limited conditions. Phosphate availability also affects significantly the production of sugars and metabolic intermediates (Hurry et al., 2000, Plant J. 24:383-396) and the lipid composition in leaves and roots (Hartel et al., 2000, Proc. Natl. Acad. Sci. USA 97:10649-10654). Likewise, the activity of the plant ACCase has been demonstrated to be regulated by phosphorylation (Savage & Ohlrogge, 1999, Plant J. 18:521-527) and alterations in the activity of the kinases and phosphatases (LMPs) that act on the ACCase could lead to increased or decreased levels of seed lipid accumulation. Moreover, the presence of lipid kinase activities in chloroplast envelope membranes suggests that signal transduction pathways and/or membrane protein regulation occur in envelopes (see, e.g., Miller et al., 2000, J. Biol. Chem. 275:19475-19481 and literature cited therein). The ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C, which are regulators in abscisic acid signaling pathway, and thereby in early and late seed development (e.g. Merlot et al., 2001, Plant J. 25:295-303). For more examples, see also the "Background of the Invention" section above.
The present invention also provides antibodies which specifically bind to an LMP polypeptide, or a portion thereof as encoded by a nucleic acid disclosed herein or as described herein.
Antibodies can be made by many well-known methods (see, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced (see, for example, Kelly et al., 1992, Bio/Technology 10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175).
The phrase "selectively binds" with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
In some instances, it is desirable to prepare monoclonal antibodies from various hosts. A description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, "Basic and Clinical Immunology," (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane ("Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York, 1988).
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and Examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims included herein.
a) General Cloning Processes:
Cloning processes such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linkage of DNA fragments, transformation of Escherichia coli and yeast cells, growth of bacteria and sequence analysis of recombinant DNA were carried out as described in Sambrook et al. (1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) or Kaiser, Michaelis and Mitchell (1994, "Methods in Yeast Genetics," Cold Spring Harbor Laboratory Press: ISBN 0-87969-451-3).
The chemicals used were obtained, if not mentioned otherwise in the text, in p.a. quality from the companies Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg), and Sigma (Deisenhofen). Solutions were prepared using purified, pyrogen-free water, designated as H2O in the following text, from a MILLI-Q water system water purification plant (Millipore, Eschborn). Restriction endonucleases, DNA-modifying enzymes, and molecular biology kits were obtained from the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Gottingen), Boehringer (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach/Taunus), Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg), Qiagen (Hilden), and Stratagene (Amsterdam, Netherlands). They were used, if not mentioned otherwise, according to the manufacturer's instructions.
c) Plant Material:
Seeds were obtained from the Arabidopsis stock center. The ecotypes Columbia 0 and Landsberg erecta were used to analyse developing seeds and siliques of early to mid stage (1 to 8 days after flowering) to late stage seeds and siliques (8 to 15 days after flowering).
Brassica napus Variety Westar
Brassica napus cv. Westar plants were used for this study to isolate developing seeds. Seeds were collected from developing seed pods 3-5 weeks after flowering.
Brassica napus AC Excel and Cresor Varieties
Brassica napus varieties AC Excel and Cresor were used for this study to create cDNA libraries. Seed, seed pod, flower, leaf stem, and root tissues were collected from plants that were in some cases dark-, salt-, heat-, or drought-treated. However, this study focused on the use of seed and seed pod tissues for cDNA libraries.
Helianthus annuus Variety Sigma
Helianthus annuus cv. Sigma plants were used for this study to create cDNA libraries from developing seeds.
d) Plant Growth:
Plants were grown on soil under standard conditions as described in Focks & Benning (1998, Plant Physiol. 118:91-101).
Brassica napus Variety Westar
Plants were either grown in the field or in Metromix (Scotts, Marysville, Ohio) in the greenhouse with supplementary lighting.
Brassica napus AC Excel and Cresor Varieties
Plants (AC Excel, except where mentioned) were grown in Metromix (Scotts, Marysville, Ohio) at 22° C. under a 14/10 light/dark cycle. Six seed and seed pod tissues of interest in this study were collected to create the following cDNA libraries: immature seeds, mature seeds, immature seed pods, mature seed pods, night-harvested seed pods, and Cresor variety (high erucic acid) seeds. Tissue samples were collected within specified time points for each developing tissue, and multiple samples within a time frame pooled together for eventual extraction of total RNA. Samples from immature seeds were taken between 1-25 days after anthesis (daa), mature seeds between 25-50 daa, immature seed pods between 1-15 daa, mature seed pods between 15-50 daa, night-harvested seed pods between 1-50 daa, and Cresor seeds 5-25 daa.
Plants were grown in Metromix (Scotts, Marysville, Ohio) at 25° C. in the greenhouse with supplementary lighting under a 14/10 light/dark cycle. Developing seeds were carefully removed with tweezers from the sunflowers 6-8 days, 13-16 days and 24-26 days after flowering of the first flowers on the outermost rim of the sunflower.
Total DNA Isolation from Plants
The details for the isolation of total DNA relate to the working up of one gram fresh weight of plant material.
CTAB buffer: 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0; 1.4 M NaCl; 20 mM EDTA. N-Laurylsarcosine buffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.
The plant material was triturated under liquid nitrogen in a mortar to give a fine powder and transferred to 2 ml EPPENDORF vessels. The frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) and incubated at 60° C. for one hour with continuous shaking. The homogenate obtained was distributed into two EPPENDORF vessels (2 ml) and extracted twice by shaking with the same volume of chloroform/isoamyl alcohol (24:1). For phase separation, centrifugation was carried out at 8000 g and room temperature for 15 minutes in each case. The DNA was then precipitated at -70° C. for 30 minutes using ice-cold isopropanol. The precipitated DNA was sedimented at 4° C. and 10,000 g for 30 minutes and resuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA was treated with NaCl (1.2 M final concentration) and precipitated again at -70° C. for 30 minutes using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and subsequently taken up in 50 μl of H2O+RNAse (50 mg/ml final concentration). The DNA was dissolved overnight at 4° C., and the RNAse digestion was subsequently carried out at 37° C. for 1 hour. Storage of the DNA took place at 4° C.
Isolation of Total RNA and Poly-(A)+ RNA from Plants
For the investigation of transcripts, both total RNA and poly-(A)+ RNA were isolated. RNA was isolated from siliques of Arabidopsis plants according to the following procedure:
RNA Preparation from Arabidopsis Seeds--"Hot" Extraction:
Buffers, Enzymes, and Solutions:
Phenol (for RNA)
(Phenol:choloroform 1:1; pH adjusted for RNA)
4 M LiCl, DEPC-treated
3M NaOAc, pH 5, DEPC-treated
70% ethanol (made up with DEPC-treated water)
Resuspension buffer:0.5% SDS, 10 mM Tris pH 7.5, 1 mM EDTA made up with DEPC-treated water as this solution can not be DEPC-treated
0.2M Na Borate
30 mM EDTA
30 mM EGTA
1% SDS (250 μl of 10% SDS-solution for 2.5 ml buffer)
1% Deoxycholate (25 mg for 2.5 ml buffer)
2% PVPP (insoluble--50 mg for 2.5 ml buffer)
2% PVP 40K (50 mg for 2.5 ml buffer)
10 mM DTT
100 mM β-Mercaptoethanol (fresh, handle under fume hood--use 35 μl of 14.3M solution for 5 ml buffer)
Extraction buffer was heated up to 80° C. Tissues were ground in liquid nitrogen-cooled mortar, and the tissue powder was transferred to a 1.5 ml tube. Tissues should be kept frozen until buffer is added; the sample should be transferred with a pre-cooled spatula; and the tube should be kept in liquid nitrogen at all times. Then 350 μl preheated extraction buffer was added (For 100 mg tissue, buffer volume can be as much as 500 μl for bigger samples) to tube; samples were vortexed; and the tube was heated to 80° C. for approximately 1 minute and then kept on ice. The samples were vortexed and ground additionally with electric mortar.
Proteinase K (0.15 mg/100 mg tissue) was added, and the mixture was vortexed and then kept at 37° C. for one hour.
For purification, 27 μl 2 M KCl was added to the samples. The samples were chilled on ice for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes at room temperature. The supernatant was transferred to a fresh, RNAase-free tube, and one phenol extraction was conducted, followed by a choloroform:isoamylalcohol extraction. One volume isopropanol to was added to the supernatant, and the mixture was chilled on ice for 10 minutes. RNA was pelleted by centrifugation (7000 rpm for 10 minutes at room temperature). Pellets were dissolved in 1 ml 4M LiCl solution by vortexing the mixture 10 to 15 minutes. RNA was pelleted by a 5 minute centrifugation.
The pellet was resuspended in 500 μl Resuspension buffer. Then 500 μl of phenol was added, and the mixture was vortexed. Then, 250 μl chloroform:isoamylalcohol was added; the mixture was vortexed and then centrifuged for 5 minutes. The supernatant was transferred to a fresh tube. The choloform:isoamylalcohol extraction was repeated until the interface was clear. The supernatant was transferred to a fresh tube and 1/10 volume 3 M NaOAc, pH 5 and 600 μl isopropanol were added. The mixture was kept at -20 for 20 minutes or longer. The RNA was pelleted by 10 minutes of centrifugation, and then the pellet was washed once with 70% ethanol. All remaining alcohol was removed before dissolving the pellet in 15 to 20 μl DEPC-treated water. The quantity and quality of the RNA was determined by measuring the absorbance of a 1:200 dilution at 260 nm and 280 nm. (40 μg RNA/ml=1 OD260).
The mRNA was prepared from total RNA, using the Amersham Pharmacia Biotech mRNA purification kit, which utilizes oligo(dT)-cellulose columns.
Isolation of Poly-(A)+ RNA was isolated using Dyna Beads® (Dynal, Oslo, Norway) following the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volume of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70° C.
Seeds were separated from pods to create homogeneous materials for seed and seed pod cDNA libraries. Tissues were ground into fine powder under liquid nitrogen using a mortar and pestle and transferred to a 50 ml tube. Tissue samples were stored at -80° C. until extractions could be performed. Total RNA was extracted from tissues using an RNEASY extraction Maxi kit (Qiagen) according to the manufacturer's protocol, and mRNA was processed from total RNA using an OLIGOTEX mRNA Purification System kit (Qiagen), also according to the manufacturer's protocol. The mRNA was sent to Hyseq Pharmaceuticals Incorporated (Sunnyville, Calif.) for further processing of mRNA from each tissue type into cDNA libraries and for use in their proprietary processes in which similar inserts in plasmids are clustered based on hybridization patterns.
Seeds were ground into fine powder under liquid nitrogen using a mortar and pestle and transferred to a 50 ml tube. Tissue samples were stored at -80° C. until extractions could be performed. Total RNA was extracted from tissues using an RNEASY RNA extraction Maxi kit (Qiagen) according to the manufacturer's protocol, and mRNA was processed from total RNA using an OLIGOTEX mRNA Purification System kit (Qiagen), also according to manufacturer's protocol. The mRNA was sent to Hyseq Pharmaceuticals Incorporated (Sunnyville, Calif.) for further processing of mRNA from each seed development stage into cDNA libraries and for use in their proprietary processes in which similar inserts in plasmids are clustered based on hybridization patterns.
cDNA Library Construction
For cDNA library construction, first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours), 16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped by incubation at 65° C. (10 minutes) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotides were removed by phenol/chloroform extraction and SEPHADEX G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated by incubation with polynucleotide kinase (Roche, 37° C., 30 minutes). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on ELUTIP-D-columns (Schleicher and Schuell, Dassel, Germany) and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the GIGAPACK Gold Kit (Stratagene, Amsterdam, Netherlands) using the material and following the instructions of the manufacturer.
Brassica and sunflower cDNA libraries were generated at Hyseq Pharmaceuticals Incorporated (Sunnyville, Calif.). No amplification steps were used in the library production to retain expression information. Hyseq's genomic approach involves grouping the genes into clusters and then sequencing representative members from each cluster. The cDNA libraries were generated from oligo dT column purified mRNA. Colonies from transformation of the cDNA library into E. coli were randomly picked and the cDNA inserts were amplified by PCR and spotted on nylon membranes. A set of 33P radiolabeled oligonucleotides were hybridized to the clones, and the resulting hybridization pattern determined to which cluster a particular clone belonged. The cDNA clones and their DNA sequences were obtained for use in overexpression in transgenic plants and in other molecular biology processes described herein.
Identification of LMP Genes of Interest
To identify potential gene targets from Arabidopsis, the MEGASORT and MPSS technologies of Lynx Therapeutics Inc. were used. MEGASORT is a micro-bead technology that allows both the simultaneous collection of millions of clones on as many micro-beads (See Brenner et al., 1999, Proc. Natl. Acad. Sci. USA 97:1665-1670). Genes were identified based on their differential expression in different developmental stages of Arabidopsis seeds and siliques. RNA and mRNA were isolated from wild-type and mutant roots using standard procedures. The MEGASORT technology enabled the identification of over- and under-expressed clones in two mRNA samples without prior knowledge of the genes and was thus useful to discover differentially expressed genes that encode LMP proteins. The MPSS technology enabled the quantitation of the abundance of mRNA transcripts in the mRNA samples (see, e.g., Brenner et al., Nat. Biotechnol. 18:630-4) and was used to obtain expression profiles of different developmental seed stages.
RNA expression profile data were obtained from the Hyseq clustering process. Clones showing 75% or greater expression in seed libraries compared to the other tissue libraries were selected as LMP candidate genes. The Brassica napus clones were selected based on their expression profile. Homologous sequences from Arabidopsis were identified using BLAST and FASTA searches and the corresponding LMPs (SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59) were isolated from Arabidopsis cDNA (See Examples 7 and 10 below).
RNA expression profile data were obtained from the Hyseq clustering process. The Helianthus annuus clones were selected for overexpression in Arabidopsis based on their predicted motifs and domains following PFAM and BLOCKS analysis of the sequences.
Identification of Proteins Associated with Oil Bodies and Microsomes in Developing Arabidopsis and Canola Seed
Isolation of Oil Bodies and Microsomes from Developing Oil Seeds
Buffers and Solutions:
1. extraction buffer
150 mM Tris pH 7.5
10 mM KCl
1.5 mM EDTA
0.1 mM MgCl2
15% w/v sucrose
2. centrifugation buffer
50 mM Tris pH 7.5
10 mM KCl
1.5 mM EDTA
0.1 mM MgCl2
10% w/v sucrose
3. gradient buffers (10, 19, 38 or 52)
50 mM Tris pH 7.5
10 mM KCl
1.5 mM EDTA
0.1 mM MgCl2
plus either of the following:
10% w/v sucrose or
19% w/v sucrose or
38% w/v sucrose or
52% w/v sucrose or
Totally green siliques of Arabidopsis thaliana or Brassica napus were opened on ice using razor blades and all developing seeds of bright green color removed carefully and placed in the pre-cooled extraction buffer. Only seeds of bright green color without brown or yellow spots, and solid consistency (in contrast to the youngest seeds) were used. The seeds were homogenized using a standard household homogenizer, razor blades, a chopping block and a glass homogenizer. The procedure was carried out on ice in the cold with frequent stops to prevent warming of the homogenate. For the smaller Arabidopsis seeds, a glass homogenizer was used immediately.
The homogenate was filtered through 2 layers of Miracloth (Calbiochem, Calif., USA) using precooled equipment. The filtered homogenate was then transferred into centrifuge tubes for an SW28 rotor, tarred, and centrifuged for 45 minutes at 8,000 rpm in a Beckman Ultrafuge (Beckman Coulter, Fullerton, Calif., USA) at 4° C. The pellet was discarded; all the liquid and the oil body layer from the top were homogenised again, transferred into new centrifuge tubes, and spun in the same ultracentrifuge for 1 hour at 4° C. and 25,000 rpm.
The resulting layer of lipid bodies was removed from the top, resuspended in centrifugation buffer, homogenized with a glass homogenizer and centrifuged again at 25,000 rpm for 1 hour at 4° C. This procedure was repeated until the oil bodies had a creamy white color. They were then removed and frozen until lipid extraction.
The pellet resulting from the first centrifugation at 25,000 rpm was kept, and resuspended in 4 ml gradient buffer containing 52% sucrose. The suspension was transferred to the bottom of a centrifuge tube for an SW28 rotor. A 10 ml gradient buffer containing 38% sucrose was carefully layered on the suspension and then topped by a layer of gradient buffer containing 19% sucrose, which was topped by a layer of gradient buffer containing 10% sucrose. An ultracentrifugation at 4° C. and 25,000 rpm was performed overnight. The centrifugation was stopped without a brake to avoid disturbances of the interfaces and layers. At the interface of the different layers, white to cream colored layers of microsomes and proteins were removed with a syringe, diluted with centrifugation buffer, and pelleted by centrifugation. The pellets were either used immediately or frozen until lipid extraction.
Lipid Extraction from Oil Bodies and Microsomes
100 mM Tris saturated Phenol (Aquaphenol) pH 8.0
100 mM Tris buffer pH 8.0
0.1 M NH4 Acetat in MeOH
Equal volumes of the Tris buffer pH 8 and the phenol (Tris saturated, pH 8) were mixed. Immediately before the lipid extraction, 5% (v/v) mercaptoethanol was added to the Tris-phenol mix. Per 100 mg of frozen or fresh sample (oil bodies or microsomes, 1 ml of mercaptoethanol-Tris-phenol was added to the sample, and the samples mixed well for 1 hour (stirrer, rotary shaker) at 4° C. An equal volume of Tris pH 8.0 buffer was added and mixed again for 30 minutes at 4° C. Then the mix was centrifuged at approximately 1,000 g at 4° C. for 15 minutes. The bottom layer (i.e. the Phenol layer) was transferred into a new 50 ml vessel without taking anything of the interface, using a syringe or pipette. Then 4 volumes of ammonium acetate in methanol was added, and the sample was precipitated by freezing at 20° C. overnight. After precipitation, the sample was centrifuged (4° C., approx 1,000 g, 30 minutes) and the pellet was kept. The pellet was washed two times with 20 ml ammonium acetate in methanol (centrifugation as before). For oil bodies, the resulting pellet was extracted with Tris-phenol-mercaptoethanol a second time; for microsomes, the pellet was directly washed 3 times with 10 ml methanol (centrifugation as before). The resulting pellet was stored with a little methanol at 4° C. until the determination of protein amount and 2D gel electrophoresis.
Determination of Protein Amount
The amount of protein was determined using the Protein Assay Kit (Sigma). According to the manufacturer's protocol, the protein samples were precipitated with TCA.
Isoelectric Focussing (IEF)--1st Dimension
For the rehydration of the IEF-strips (e.g. IMMOBILINE DryStrip pH 4-7, 24 cm, from Amersham), freshly prepared buffer is used:
14.41 g (8 M) urea
4.57 g (2 M) Thiourea
92.52 mg (20 mM) Dithiothreitol (DTT)
300 mg (1%) CHAPS detergent
156 μl Ampholine (Amersham) pH 3.5-10
fill up to 30 ml with ddH2O
Typically 400-1500 μg protein were used for a preparative 2D gel with Coomassie staining. The required amount of protein in solution was diluted with the rehydration buffer to a total volume of 600 μl and vortexed well. The solution was spread along the groove of the IMMOBILINE DryStrip Reswelling Trays (Amersham). The protective cover of the IEF strip was removed, and the strip placed carefully into the tray with the gel side down, making sure that the strip had good contact with the sample solution and no air bubbles were trapped between the solution and the strip. When loading of the strips was complete, the chamber was sealed (to protect from evaporation) and left at ambient temperature for 24 hours.
After 24 hours, the strips were taken from the chamber, padded slightly dry with wet WHATMAN filter paper No. 3 and placed into the "IMMOBILINE strip tray" of the MIULTIPHOR chamber (Pharmacia) with the gel side up. Then, 90 to 100 ml cover fluid (DryStrip Cover Fluid, Amersham) was added. Two electrode strips were being soaked with dH2O, and the surplus water was removed with a paper tissue. On the cathode as well as on the anode side of the strips, one of the electrode strips was placed across the gel strips. The electrode chamber was closed with the electrode in place, and the focusing was performed according to the procedure given in Table 5.
TABLE-US-00005 TABLE 5 Parameter for the isoelectric focusing voltage current power Voltage (V) (mA) (W) pattern hours (Vh) 500 1 5 Gradient 500 500 1 5 Gradient 2500 3500 1 5 Gradient 10.000 3500 1 5 Gradient 45.000
At the end of the focusing, the current was interrupted, the chamber was opened, and the gel strips were padded slightly on wetted tissue to remove cover fluid. The gel strips could be used immediately, or if necessary, the gel strips could be stored at 80° C. until further use.
SDS-Polyacrylamide Gel Electrophoresis (PAGE)--2nd Dimension
Preparation of the Ettan DALT-II Electrophoresis Chamber
The electrophoresis chamber was first filled with 7.5 1 of dH2O. The control unit and the pump were switched on, and 75 ml of the concentrated anode buffer was added (ETTAN DALT II Buffer Kit, Amersham). The 2D gels (pre-cast ETTAN DALT II Gel (12.5%), Amersham) and 2 ml gel buffer were loaded into the gel frame; the surplus gel buffer was removed with a common wallpaper roller; and the frame was closed. Surplus buffer was removed by tilting the frame. The left and right side of the frame were sealed with agarose melted at 85° C. The bottom end of the frame was wetted with dH2O and the frame was inserted into the ETTAN DALT chamber. The cathode buffer (ETTAN DALT II Buffer Kit,. Amersham), diluted 1:10, was used to fill the chamber up to the mark.
Equilibration of the Gel Strips
Equilibration stock buffer: 36 g (6 M) urea 30 g (30%) glycerol 2 g (2%) SDS 3.3 ml Tris-HCl-buffer pH 8.8 (18.2 g (1.5 M) Tris/HCl, 0.4 g (0.4%) SDS, pH 8.8 ad 100 ml with dH2O) add 100 ml with dH2ODTT equilibration buffer (per gel strip): 4 ml Equilibration stock buffer 20 μl Bromophenolblue solution (30 mg Bromophenolblue in 10 ml Tris/HCl-buffer pH 8.8) 200 μl DTT stock solution (200 mg DTT+1 ml dH2O)Jodacetamide equilibration buffer (per gel strip): 4 ml Equilibration stock buffer 20 μl Bromophenolblue solution 192 mg (260 μM) Jodacetamid
The gel strip was placed into the equilibration tray with the gel side up. First, 4 ml of the DTT equilibration buffer was added, and the tray was shaken for 15 minutes on a rotary shaker. The buffer was discarded, and the strip was shaken for 15 minutes with the Jodacetamide equilibration buffer. The buffer was again removed by decanting, and the surplus equilibration buffer was removed by patting the gel strips on wetted tissue.
The gel strips were orientated with the support side toward the glass slide of the gel, and inserted into the groove between the glass slide of the gel frame and the support of the gel. After moving the strip down and into contact with the gel, the strip was pressed slightly against the gel. If necessary, air bubbles were removed. The chamber was closed, and the electrophoretic run was performed according to the parameters given in table 6:
TABLE-US-00006 TABLE 6 Parameter for the SDS-PAGE in the ETTAN-Dalt-chamber Power per Temp. time step pump gel (W) (° C.) (min) comments 1 Auto 4 25 75 const. Power 2 Auto 14 25 360 const. Power
When the dye front reached the bottom end of the chamber, the current was broken and the chamber opened. The gels were removed from the gel frames and stored in the fixing solution (see staining of the gel) under constant shaking for a period of no less then 2 hours, usually overnight.
Coomassie Staining of the Gels
Per gel, 500 ml of the reagent solution was needed. The gels were shaken on a rotary shaker at 40 rpm. Table 7 summarizes the steps involved:
20 ml (2%, v/v) phosphoric acid (85%)
100 g (10%, w/v) Ammonium sulphate
200 ml (20%, v/v) Methanol
1 g (0.1%) SERVA Blue C, (Serva, Germany)
add dH2O to 1000 ml
TABLE-US-00007 TABLE 7 Coomassie-staining of the proteins after the 2D-PAGE Step Solution Incubation period Fixation 40% (v/v) Methanol >2 h 10% (v/v) acetic acid 50% (v/v) deionised H2O Washing Deionized H2O --H2O 20 minutes Washing Deionized H2O --H2O 20 minutes Washing Deionized H2O --H2O 20 minutes Aequilibration 2% (v/v) Phosphoric acid 2 hours 10% (v/v) Methanol staining Satining solution (see below) 12-24 hours washing Deionized H2O --H2O 30 minutes
Identification of Proteins Separated by 2D Gel Electrophoresis.
Protein spots were excised from Coomassie blue-stained gels, washed with MeOH/H2O and CAN, and digested with trypsin (Roche, Mannheim) overnight at 37° C. For nanoHPLC/MS/MS analysis, the peptides were separated using reverse phase chromatography (RP18-3, 100 Å, 15 cm, 75 μm i.d., from LC-Packings). Peptide analysis was performed on a LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose). A mass spectrum in full-scan mode was followed by two MS/MS spectra of the most abundant peptide ions. Peptide tandem mass spectra were analysed using the MASCOT software package (Matrix Science, Ltd, London, UK), and peptides matched to publicly available databases of Arabidopsis proteins. Proteins identified in this manner were analyzed for domains possibly involved in regulation of metabolic processes. For selected proteins (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12), the full-length cDNA (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11) was cloned as described above.
Cloning Full-Length cDNAs and Orthologs of Identified LMP Genes Arabidopsis thaliana
Full-length sequences of the Arabidopsis thaliana partial cDNAs (ESTs) that were identified from MEGASORT and MPSS EST sequencing were isolated by RACE PCR using the SMART RACE cDNA amplification kit from Clontech allowing both 5' and 3' rapid amplification of cDNA ends (RACE). The isolation of cDNAs and the RACE PCR protocol used were based on the manufacturer's conditions. The RACE product fragments were extracted from agarose gels with a QIAQUICK Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into TOP10 cells (Invitrogen) using standard conditions (Sambrook et al., 1989). Transformed cells were grown overnight at 37° C. on LB agar containing 50 μg/ml kanamycin and spread with 40 μl of a 40 mg/ml stock solution of X-gal in dimethylformamide for blue-white selection. Single white colonies were selected and used to inoculate 3 ml of liquid LB containing 50 μg/ml kanamycin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAPREP Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Subsequent analyses of clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al., 1989).
Gene sequences can be used to identify homologous or heterologous genes (orthologs, the same LMP gene from another plant) from cDNA or genomic libraries. This can be done by designing PCR primers to conserved sequences identified by multiple sequence alignments. Orthologs are often identified by designing degenerate primers to hill-length or partial sequences of genes of interest. Homologous genes (e.g. full-length cDNA clones) can be isolated via nucleic acid hybridization using, for example, cDNA libraries: Depending on the abundance of the gene of interest, 100,000 up to 1,000,000 recombinant bacteriophages are plated and transferred to nylon membranes. After denaturation with alkali, DNA is immobilized on the membrane by e.g. UV cross linking. Hybridization is carried out at high stringency conditions. Aqueous solution hybridization and washing is performed at an ionic strength of 1 M NaCl and a temperature of 68° C. Hybridization probes are generated by, e.g., radioactive (32P) nick transcription labeling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.
Partially homologous or heterologous genes that are related but not identical can be identified in a procedure analogous to the above-described procedure using low stringency hybridization and washing conditions. For aqueous hybridization, the ionic strength is normally kept at 1 M NaCl while the temperature is progressively lowered from 68 to 42° C.
Isolation of gene sequences with homology (or sequence identity/similarity) only in a distinct domain (for example, 10-20 amino acids) can be carried out by using synthetic radiolabeled oligonucleotide probes. Radiolabeled oligonucleotides are prepared by phosphorylation of the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are than radiolabeled by, for example, nick transcription. Hybridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
Oligonucleotide Hybridization Solution:
0.01 M sodium phosphate
1 mM EDTA (pH 8)
100 μg/ml denaturated salmon sperm DNA0.1% nonfat dried milk
During hybridization, temperature is lowered stepwise to 5-10° C. below the estimated oligonucleotide Tm or down to room temperature followed by washing steps and autoradiography. Washing is performed with low stringency such as three washing steps using 4×SSC. Further details are described by Sambrook et al. (1989, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press) or Ausubel et al. (1994, "Current Protocols in Molecular Biology," John Wiley & Sons).
Clones of Helianthus annuus genes obtained from Hyseq were sequenced at using a ABI 377 slab gel sequencer and BIGDYE Terminator Ready Reaction kits (PE Biosystems, Foster City, Calif.). The isolation of cDNAs and the RACE PCR protocol used were based on the manufacturer's conditions. The RACE product fragments were extracted from agarose gels with a QIAQUICK Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into TOP10 cells (Invitrogen) using standard conditions (Sambrook et al., 1989). Transformed cells were grown overnight at 37° C. on LB agar containing 50 μg/ml kanamycin and spread with 40 μl of a 40 mg/ml stock solution of X-gal in dimethylformamide for blue-white selection. Single white colonies were selected and used to inoculate 3 ml of liquid LB containing 50 μg/ml kanamycin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAPREP Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Subsequent analyses of clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al., 1989).
RT-PCR and Cloning of Arabidopsis thaliana and Helianthus annuus LMP Genes
Full-length LMP cDNAs were isolated by RT-PCR from Arabidopsis thaliana or Helianthus annuus RNA. The synthesis of the first strand cDNA was achieved using AMV Reverse Transcriptase (Roche, Mannheim, Germany). The resulting single-stranded cDNA was amplified via Polymerase Chain Reaction (PCR) utilizing two gene-specific primers. The conditions for the reaction were standard conditions with Expand High Fidelity PCR system (Roche). The parameters for the reaction were: five minutes at 94° C. followed by five cycles of 40 seconds at 94° C., 40 seconds at 50° C., and 1.5 minutes at 72° C. This was followed by thirty cycles of 40 seconds at 94° C., 40 seconds at 65° C., and 1.5 minutes at 72° C. The fragments generated under these RT-PCR conditions were analyzed by agarose gel electrophoresis to make sure that PCR products of the expected length had been obtained.
Full-length LMP cDNAs were isolated by using synthetic oligonucleotide primers (MWG-Biotech) designed based on the LMP gene specific DNA sequence that was determined by EST sequencing and by sequencing of RACE PCR products. The 5' PCR primers ("forward primer," F) SEQ ID NO:61, SEQ ID NO:63, SEQ ID N0:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, and SEQ ID NO:119 (for amplification of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:121, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59, respectively) contained an NotI restriction site 5' upstream of the ATG start codon. The 5' PCR primer ("forward primer," F) SEQ ID NO:95 for amplification of SEQ ID NO:11 contained PstI and NotI restriction sites 5' upstream of the ATG start codon. The 3' PCR primers ("reverse primer," R) SEQ ID NO:62 and SEQ ID NO:92 (for amplification of SEQ ID NO:23 and SEQ ID NO:7, respectively) contained an EcoRV restriction site 3' downstream of the stop codon. The 3' PCR primer ("reverse primer," R) SEQ ID NO:64 for amplification of SEQ ID NO:25 contained a SmaI restriction site 3' downstream of the stop codon. The 3' PCR primers ("reverse primer," R) SEQ ID NO:66, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:1108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, and SEQ ID NO:120 (for amplification of SEQ ID NO:27, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:121, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59, respectively) contained a StuI restriction site 3' downstream of the stop codon. The 3' PCR primers ("reverse primer," R) SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, and SEQ ID NO:84 (for amplification of SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45, respectively) contained a NotI restriction site 3' downstream of the stop codon.
The restriction sites were added so that the RT-PCR amplification products could be cloned into the restriction sites located in the multiple cloning site of the binary vector. The following "forward" (F) and "reverse" (R) primers were used to amplify the full-length Arabidopsis thaliana or Helianthus annuus cDNAs by RT-PCR using RNA from Arabidopsis thaliana or Helianthus annuus as original template:
TABLE-US-00008 For amplification of SEQ ID NO: 1: Osw14F (5'- GCGGCCGCCATGGCAACGGGGGCTGAGAACC -3') (SEQ ID NO: 85) Osw14R (5'- AGGCCTTTACCGGCGACCACCAGCAGG -3') (SEQ ID NO: 86) For amplification of SEQ ID NO: 3: Osw15F (5'- GCGGCCGCCATGGCAACCATGGCTAGGTCG -3') (SEQ ID NO: 87) Osw15R (5'- AGGCCTTCAGTTGTCGTGCAATGCTTTG -3') (SEQ ID NO: 88) For amplification of SEQ ID NO: 5: Osw16F (5'- GCGGCCGCCATGGCGCAACCCCTCGTGAAG -3') (SEQ ID NO: 89) Osw16R (5'- AGGCCTTTAGCCGCTGGCAACAATCTC -3') (SEQ ID NO: 90) For amplification of SEQ ID NO: 7: Osw17F (5'- GCGGCCGCCATGCCGATTAGCCGGAGAGTTC -3') (SEQ ID NO: 91) Osw17R (5'- GATATCTCATATGGAATCATAAACCG -3') (SEQ ID NO: 92) For amplification of SEQ ID NO: 9: Qsw18F (5'- GCGGCCGCCATGCCCATTAATCAGAAAATTC -3') (SEQ ID NO: 93) Osw18R (5'- AGGCCTTCATTTGCGATCAAGAACC -3') (SEQ ID NO: 94) For amplification of SEQ ID NO: 11: Osw20F (5'- CTGCAGGCGGCCGCCATGGAGTGTAGTTCAGTGAGTG -3') (SEQ ID NO: 95) Osw20R (5'- AGGCCTCTAGTATTGGACTAACGATAAC -3') (SEQ ID NO: 96) For amplification of SEQ ID NO: 13: Osw21F (5'- GCGGCCGCATGGTTGAAACCTTGTTTGAAG -3') (SEQ ID NO: 97) Osw21R (5'- AGGCCTCTAGGCCTTATCCACCTTCC -3') (SEQ ID NO: 98) For amplification of SEQ ID NO: 15: Osw22F (5'- GCGGCCGCATGGGCAAAAAAGACATGCCTA -3') (SEQ ID NO: 99) Osw22R (5'- AGGCCTTCATATTGTCGTGTAACGAGGG -3') (SEQ ID NO: 100) For amplification of SEQ ID NO: 17: Osw23F (5'- GCGGCCGCATGATATTATCGTTTCGTGGAC -3') (SEQ ID NO: 101) Osw23R (5'- AGGCCTCTATACAGATTTGCCATCGCTC -3') (SEQ ID NO: 102) For amplification of SEQ ID NO: 19: Osw24F (5'- GCGGCCGCATGAACCGGATGATCGAAGCG -3') (SEQ ID NO: 103) Osw24R (5'- AGGCCTTCATATTTGGTGCACCTCGGC -3') (SEQ ID NO: 104) For amplification of SEQ ID NO: 21 or SEQ ID NO: 121: Osw26F (5'- GCGGCCGCATGCAGACCGTTTCTCGGAG -3') (SEQ ID NO: 105) Osw26R (5'- AGGCCTTCAAGGATAAGACTCTGGAG -3') (SEQ ID NO: 106) For amplification of SEQ ID NO: 23: JB69F (5'- GCGGCCGCCATGAAGATATACTCTAGAACG -3') (SEQ ID NO: 61) JB69R (5'- GATATCTTAGGCTGCCTCTGCAAACCC -3') (SEQ ID NO: 62) For amplification of SEQ ID NO: 25: JB70F (5'- GCGGCCGCCATGGGAGTATACTCGAGAGCG -3') (SEQ ID NO: 63) JB70R (5'- CCCGGGTCACACGGCTTCTGCGAAGCC -3') (SEQ ID NO: 64) For amplification of SEQ ID NO: 27: JB71F (5'- GCGGCCGCCATGGGAACTAGGTTCCAATCA -3') (SEQ ID NO: 65) JB71R (5'- AGGCCTTTAAGCAGCTTTGGCGAATCC -3') (SEQ ID NO: 66) For amplification of SEQ ID NO: 29: JB80F (5'- GAGCGGCCGCCATGGAGAAAGGTTTGACGAT -3') (SEQ ID NO: 67) JB80R (5'- GAGCGGCCGCCTTAAGGATGCAAGGGCTCCT -3') (SEQ ID NO: 68) For amplification of SEQ ID NO: 31: JB82F (5'- GAGCGGCCGCCATGCATTGGCATGGTGTAGAGCAG -3') (SEQ ID NO: 69) JB82R (5'- GAGCGGCCGCCTTATTCATAGCAAGGCGGCA -3') (SEQ ID NO: 70) For amplification of SEQ ID NO: 33: JB84F (5'- GAGCGGCCGCCATGTCTGCTTCTGATTCCTCT -3') (SEQ ID NO: 71) JB84R (5'- GAGCGGCCGCCTTAGTCGCGGAACTCGTCCA -3') (SEQ ID NO: 72) For amplification of SEQ ID NO: 35: JB85F (5'- GAGCGGCCGCCATGGCGTTCCCTAAGGTATACTT -3') (SEQ ID NO: 73) JB85R (5'- GAGCGGCCGCCCTAAGAGAGCTGACCACAAT -3') (SEQ ID NO: 74) For amplification of SEQ ID NO: 37: JB88F (5'- GAGCGGCCGCCATGGGTAGTGCAAAATCAGC -3') (SEQ ID NO: 75) JB88R (5'- GAGCGGCCGCCTTAGGCGATGGAGCTTTTAT -3') (SEQ ID NO: 76) For amplification of SEQ ID NO: 39: JB89F (5'- GAGCGGCCGCCATGACTAATCCCATGATCAT -3') (SEQ ID NO: 77) JB89R (5'- GAGCGGCCGCCCTAGAGACGGTGGATCAACG -3') (SEQ ID NO: 78) For amplification of SEQ ID NO: 41: JB90F (5'- GAGCGGCCGCCATGGTTTCTTCTTCTTTAAC -3') (SEQ ID NO: 79) JB90R (5'- GAGCGGCCGCCTTAATAATTGGTAGCTTTAT -3') (SEQ ID NO: 80) For amplification of SEQ ID NO: 43: JB91F (5'- GAGCGGCCGCCATGGCCGGAGTTTTCAAAAC -3') (SEQ ID NO: 81) JB91R (5'- GAGCGGCCGCCTCAAAAGAGAGCAACAACAG -3') (SEQ ID NO: 82) For amplification of SEQ ID NO: 45: JB93F (5'- GAGCGGCCGCCATGGCGTCAAAGCAACTGAG -3') (SEQ ID NO: 83) JB93R (5'- GAGCGGCCGCCTCACTTGTTGGTGAACTTTG -3') (SEQ ID NO: 84) For amplification of SEQ ID NO: 47: ToZ01F (5'- GCGGCCGCATGGCTTCGGTTACTTTCTCT -3') (SEQ ID NO: 107) ToZ01R (5'- AGGCCTTCACTTCCAGTTGTTGGCAA -3') (SEQ ID NO: 108) For amplification of SEQ ID NO: 49: ToZ02F (5'- GCGGCCGCATGGCAAAAGAAAATGGATT -3') (SEQ ID NO: 109) ToZ02R (5'- AGGCCTTTAGATAGAGAGGTCAGCGA -3') (SEQ ID NO: 110) For amplification of SEQ ID NO: 51: ToZ03F (5'- GCGGCCGCATGGCGGCGAAAATTCCCGG -3') (SEQ ID NO: 111) ToZ03R (5'- AGGCCTTCAAGACATGAACAGAGCCT -3') (SEQ ID NO: 112) For amplification of SEQ ID NO: 53: ToZ04F (5'- GCGGCCGCATGGGTTACATAGGAGCTCAT -3') (SEQ ID NO: 113) ToZ04R (5'- AGGCCTTCAAGCTTCTTTACGCGTGA -3') (SEQ ID NO: 114) For amplification of SEQ ID NO: 55: ToZ05F (5'- GCGGCCGCATGTCTCCTTCTCACTCCATCA -3') (SEQ ID NO: 115) ToZ05R (5'- AGGCCTTCATTTGGTGTTTGAAATAT -3') (SEQ ID NO: 116) For amplification of SEQ ID NO: 57: ToZ11F (5'- GCGGCCGCATGGCATCTGTTTACTCCACCCTA -3') (SEQ ID NO: 117) ToZ11R (5'- AGGCCTTTAATCGTTTTTCTTGGAAA -3') (SEQ ID NO: 118) For amplification of SEQ ID NO: 59: ToZ12F (5'- GCGGCCGCATGGCATCAATTTACTCCTCTTT -3') (SEQ ID NO: 119) ToZ12R (5'- AGGCCTCTAATCGCTTTTTTTGCCAT -3') (SEQ ID NO: 120)
Identification of Genes of Interest by Screening Expression Libraries with Antibodies
The cDNA clones can be used to produce recombinant protein, for example, in E. coli (e.g. Qiagen QIAEXPRESS pQE system). Recombinant proteins are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant proteins can be used to produce specific antibodies, for example, by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Cu et al. (1994, BioTechniques 17:257-262). The antibody can then be used to screen expression cDNA libraries to identify homologous or heterologous genes via an immunological screening (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press; or Ausubel et al. 1994, "Current Protocols in Molecular Biology", John Wiley & Sons).
For RNA hybridization, 20 μg of total RNA or 1 μg of poly-(A)+ RNA was separated by gel electrophoresis in 1.25% strength agarose gels using formaldehyde as described in Amasino (1986, Anal. Biochem. 152:304), transferred by capillary attraction using 10×SSC to positively charged nylon membranes (HYBOND N+, Amersham, Braunschweig), immobilized by UV light, and pre-hybridized for 3 hours at 68° C. using hybridization buffer (10% dextran sulfate w/v, 1 M NaCl, 1% SDS, 100 μg/ml of herring sperm DNA). The labeling of the DNA probe with the Highprime DNA labeling kit (Roche, Mannheim, Germany) was carried out during the pre-hybridization using α-32P dCTP (Amersham, Braunschweig, Germany). Hybridization was carried out after addition of the labeled DNA probe in the same buffer at 68° C. overnight. The washing steps were carried out twice for 15 minutes using 2×SSC and twice for 30 minutes using 1×SSC, 1% SDS at 68° C. The exposure of the sealed filters was carried out at -70° C. for a period of 1 day to 14 days.
Computational Functional Analysis
Sequences were processed using the software packages GENOMAX and VECTOR NTI (both commercially provided by Informax, Frederick, Md., USA) and annotated using the software packages GENOMAX and PEDANT-PRO commercially provided by Bio-Max (Munich, Germany). The programs incorporate practically all bioinformatics methods important for functional and structural characterization of protein sequences. For reference, see http://www.informaxinc.com/ and http://pedant.mips.biochem.mpg.de.
The most important algorithms incorporated in GENOMAX and PEDANT-PRO are: FASTA: Very sensitive protein sequence database searches with estimates of statistical significance (Pearson W. R., 1990, Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 183:63-98); BLAST: Very sensitive protein sequence database searches with estimates of statistical significance (Altschul S. F. et al., Basic local alignment search tool. J. Mol. Biol. 215:403-410); PREDATOR: High-accuracy secondary structure prediction from single and multiple sequences (Frishman & Argos 1997, 75% accuracy in protein secondary structure prediction. Proteins 27:329-335); CLUSTALW: Multiple sequence alignment (Thompson, J. D. et al., 1994, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22:4673-4680); TMAP: Transmembrane region prediction from multiply aligned sequences (Persson B. & Argos P. 1994, Prediction of transmembrane segments in proteins utilizing multiple sequence alignments, J. Mol. Biol. 237:182-192); ALOM2: Transmembrane region prediction from single sequences (Klein P., Kanehisa M., and DeLisi C. 1984, Prediction of protein function from sequence properties: A discriminant analysis of a database. Biochim. Biophys. Acta 787:221-226. Version 2 by Dr. K. Nakai); PROSEARCH: Detection of PROSITE protein sequence patterns (Kolakowski L. F. Jr. et al., 1992, ProSearch: fast searching of protein sequences with regular expression patterns related to protein structure and function. Biotechniques 13:919-921); BLIMPS: Similarity searches against a database of ungapped blocks (Wallace & Henikoff 1992, PATMAT:A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford); PFAM and BLOCKS searches of protein motifs and domains.
Plasmids for Plant Transformation
For plant transformation, various binary vectors such as a pSUN2 and pSUN300 plant binary vector were used. Construction of the plant binary vectors was performed by ligation of the cDNA in sense or antisense orientation into the vector. In such vectors, a plant promoter was located 5' to the cDNA, where it activated transcription of the cDNA; and a polyadenylation sequence was located 3' to the cDNA. Various plant promoters were used, such as a constitutive promoter (Superpromoter), a seed-specific promoter, and a root-specific promoter. Tissue-specific expression was achieved by using a tissue-specific promoter. For example, in some instances, seed-specific expression was achieved by cloning the napin or LeB4 or USP promoter 5-prime to the cDNA. Also, any other seed specific promoter element can be used, and such promoters are well known to one of ordinary skill in the art. For constitutive expression within the whole plant, in some instances, the Superpromoter or the CaMV 35S promoter was used. The expressed protein also can be targeted to a cellular compartment using a signal peptide, for example for plastids, mitochondria, or endoplasmic reticulum (Kermode, 1996, Crit. Rev. Plant Sci. 15:285-423). The signal peptide is cloned 5' in frame to the cDNA to achieve subcellular localization of the fusion protein.
The plant binary vectors comprised a selectable marker gene driven under the control of one of various plant promoters, such as the AtAct2-1 promoter and the Nos-promoter; the LMP candidate cDNA under the control of a root-specific promoter, a seed-specific promoter, a non-tissue specific promoter, or a constitutive promoter; and a terminator. Partial or full-length LMP cDNA was cloned into the plant binary vector in sense or antisense orientation behind the desired promoter. The recombinant vector containing the gene of interest was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing the selective agent, and cells were grown overnight at 37° C. Plasmid DNA was extracted using the QIAPREP Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analysis of subsequent clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).
Agrobacterium Mediated Plant Transformation
Agrobacterium mediated plant transformation with the LMP nucleic acids described herein can be performed using standard transformation and regeneration techniques (Gelvin, Stanton B. & Schilperoort R. A, Plant Molecular Biology Manual, 2nd ed. Kluwer Academic Publ., Dordrecht 1995 in Sect., Ringbuc Zentrale Signatur:BT11-P; Glick, Bernard R. and Thompson, John E. Methods in Plant Molecular Biology and Biotechnology, S. 360, CRC Press, Boca Raton 1993). For example, Agrobacterium mediated transformation can be performed using the GV3 (pMP90) (Koncz & Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.
Arabidopsis thaliana can be grown and transformed according to standard conditions (Bechtold, 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al., 1994, Science 265:1856-1860). Additionally, rapeseed can be transformed with the LMP nucleic acids of the present invention via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Additionally, Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al. (1994, Plant Cell Report 13:282-285).
Transformation of soybean can be performed using, for example, a technique described in EP 0424 047, U.S. Pat. No. 5,322,783 (Pioneer Hi-Bred International) or in EP 0397 687, U.S. Pat. No. 5,376,543 or U.S. Pat. No. 5,169,770 (University Toledo). Soybean seeds are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with continuous shaking. Then the seeds are rinsed four times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a sealed Petri dish until further use.
This method of plant transformation is also applicable to Brassica and other crops. In particular, seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes, at room temperature with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. The seed coats are removed and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of their water content. The seeds are then stored at room temperature in a sealed Petri dish until further use.
Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and resuspended in MS (Murashige & Skoog, 1962, Physiol. Plant. 15:473-497) medium supplemented with 100 mM acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 44% moisture content are imbibed for 2 hours at room temperature with the pre-induced Agrobacterium suspension culture. (The imbibition of dry embryos with a culture of Agrobacterium is also applicable to maize embryo axes).
The embryos are removed from the imbibition culture and are transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature. Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/l carbenicillin or 300 mg/l cefotaxime to kill the agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25° C., under 440 μmol m-2s-1 and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favor the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25° C., under 440 μmol m-2s-1 light intensity and 12 hour photoperiod for about 80 days.
Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization wherein DNA is electrophoresed on a 1% agarose gel and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labeled probe by PCR as recommended by the manufacturer.
In Vivo Mutagenesis
In vivo mutagenesis of microorganisms can be performed by incorporation and passage of the plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp W. D. 1996, DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.). Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener and Callahan, 1994, Strategies 7:32-34. Transfer of mutated DNA molecules into plants is preferably done after selection and testing in microorganisms. Transgenic plants are generated according to various examples throughout this document.
Assessment of the mRNA Expression and Activity of a Recombinant Gene Product in the Transformed Organism
The activity of a recombinant gene product in the transformed host organism can be measured on the transcriptional level or/and on the translational level. A useful method to ascertain the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information at least partially demonstrates the degree of transcription of the transformed gene. Total cellular RNA can be prepared from plant cells, tissues or organs by several methods, all well-known in the art, such as that described in Bormann et al. (1992, Mol. Microbiol. 6:317-326).
To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as a Western blot, may be employed (See, for example, Ausubel et al. 1988, Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.
The activity of LMPs that bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such LMP on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar H. et al., 1995, EMBO J. 14:3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both prokaryotic and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of lipid metabolism membrane-transport proteins can be performed according to techniques such as those described in Gennis R. B. (1989 Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137, 199-234 and 270-322).
In Vitro Analysis of the Function of Arabidopsis thaliana, Brassica Napus, and Helianthus annuus Genes in Transgenic Plants
The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one skilled in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M. & Webb, E. C., 1979, Enzymes. Longmans: London; Fersht, 1985, Enzyme Structure and Mechanism. Freeman: New York; Walsh, 1979, Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C., Stevens, L., 1982, Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3rd ed. Academic Press: New York; Bisswanger, H., 1994, Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes. VCH: Weinheim, p. 352-363.
Analysis of the Impact of Recombinant LMPs on the Production of a Desired Seed Storage Compound
Fatty Acid Production
The total fatty acid content of Arabidopsis seeds was determined by conventional gas-liquid chromatography (GLC) analyses after conversion to methyl esters (Schulte & Weber, 1989, Fat Sci. Technol. 91:181). For this, total fatty acids were extracted with methyl tertiary-butyl ether (MTBE) and derivatized to their corresponding fatty acid methyl esters (FAMEs) with trimethylsulfonium hydroxide (TMSH). The separation of the FAMEs was performed on a capillary column (DB Wax 10 m×0.1 mm×0.2 μm) in an Agilent Technology 6890N Network GC System equipped with a flame ionisation detector (FID) using hydrogen as carrier gas. Determination of the fatty acid content was performed by adding an internal standard of known concentration. The total fatty acid contents of seeds of transgenic plants and plants containing the pBPS empty vector construct (without an LMP gene of interest) were measured. Bulked seeds (usually 5 mg seed weight) of a single plant were used; all extractions were performed in duplicate or triplicate. The controls indicated in the tables below have been grown side by side with the transgenic lines. Differences in the total values of the controls are explained either by differences in the growth conditions, which were found to be very sensitive to small variations in the plant cultivation, or by differences in the standards added to quantify the fatty acid content. Because of the seed bulking, all values obtained with T2 seeds, and in part also with T3 seeds, are the result of a mixture of homozygous (for the gene of interest) and heterozygous events, implying that these data underestimate the LMP gene effect.
TABLE-US-00009 TABLE 8 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW14 (containing SEQ ID NO: 1). Shown are the means (±standard deviation) of 4 individual control plants and 6 individual plants per line. Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.253 ± 0.009 OSW14 transgenic seeds 0.270 ± 0.006
TABLE-US-00010 TABLE 9 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW15 (containing SEQ ID NO: 3). Shown are the means (±standard deviation) of 3 individual control plants and 18 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.259 ± 0.008 OSW15 transgenic seeds 0.300 ± 0.010
TABLE-US-00011 TABLE 10 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW16 (containing SEQ ID NO: 5). Shown are the means (±standard deviation) of 3 individual control plants and 10 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.296 ± 0.024 OSW16 transgenic seeds 0.318 ± 0.002
TABLE-US-00012 TABLE 11 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW17 (containing SEQ ID NO: 7). Shown are the means (±standard deviation) of 3 individual control plants and 5 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.292 ± 0.008 OSW17 transgenic seeds 0.315 ± 0.004
TABLE-US-00013 TABLE 12 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW18 (containing SEQ ID NO: 9). Shown are the means (±standard deviation) of 3 individual control plants and 14 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.282 ± 0.002 OSW18 transgenic seeds 0.309 ± 0.007
TABLE-US-00014 TABLE 13 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW21 (containing SEQ ID NO: 13). Shown are the means (±standard deviation) of 4 individual control plants and 22 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.242 ± 0.025 OSW21 transgenic seeds 0.283 ± 0.016
TABLE-US-00015 TABLE 14 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb069 (containing SEQ ID NO: 23). Shown are the means (±standard deviation) of 4 individual control plants and 8 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.193 ± 0.016 JB069 transgenic sedes 0.211 ± 0.005
TABLE-US-00016 TABLE 15 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb070 (containing SEQ ID NO: 25). Shown are the means (±standard deviation) of 4 individual control plants and 7 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.193 ± 0.016 JB070 transgenic sedes 0.218 ± 0.003
TABLE-US-00017 TABLE 16 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb071 (containing SEQ ID NO: 27). Shown are the means (±standard deviation) of 4 individual control plants and 13 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.246 ± 0.009 JB071 transgenic sedes 0.278 ± 0.007
TABLE-US-00018 TABLE 17 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb080 (containing SEQ ID NO: 29). Shown are the means (±standard deviation) of 4 individual control plants and 19 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.240 ± 0.017 JB080 transgenic seeds 0.268 ± 0.006
TABLE-US-00019 TABLE 18 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb082 (containing SEQ ID NO: 31). Shown are the means (±standard deviation) of 4 individual control plants and 8 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.240 ± 0.014 JB082 transgenic seeds 0.264 ± 0.007
TABLE-US-00020 TABLE 19 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb084 (containing SEQ ID NO: 33). Shown are the means (±standard deviation) of 3 individual control plants and 8 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.379 ± 0.025 JB084 transgenic seeds 0.426 ± 0.004
TABLE-US-00021 TABLE 20 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb085 (containing SEQ ID NO: 35). Shown are the means (±standard deviation) of 4 individual control plants and 8 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.231 ± 0.004 JB085 transgenic seeds 0.263 ± 0.009
TABLE-US-00022 TABLE 21 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb088 (containing SEQ ID NO: 37). Shown are the means (±standard deviation) of 4 individual control plants and 7 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.353 ± 0.024 JB088 transgenic seeds 0.392 ± 0.016
TABLE-US-00023 TABLE 22 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb089 (containing SEQ ID NO: 39). Shown are the means (±standard deviation) of 4 individual control plants and 4 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.314 ± 0.007 JB089 transgenic seeds 0.332 ± 0.006
TABLE-US-00024 TABLE 23 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb090 (containing SEQ ID NO: 41). Shown are the means (±standard deviation) of 4 individual control plants and 4 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.321 ± 0.011 JB090 transgenic seeds 0.271 ± 0.020
TABLE-US-00025 TABLE 24 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb091 (containing SEQ ID NO: 43). Shown are the means (±standard deviation) of 4 individual control plants and 4 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.279 ± 0.015 JB091 transgenic seeds 0.307 ± 0.004
TABLE-US-00026 TABLE 25 Determination of the T2 seeds of total fatty acid content of transgenic lines of Jb093 (containing SEQ ID NO: 45). Shown are the means (±standard deviation) of 4 individual control plants and 12 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.353 ± 0.024 JB093 transgenic seeds 0.258 ± 0.024
TABLE-US-00027 TABLE 26 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW22 (containing SEQ ID NO: 15). Shown are the means (±standard deviation) of 4 individual control plants and 5 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.272 ± 0.025 OSW22 transgenic seeds 0.291 ± 0.006
TABLE-US-00028 TABLE 27 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW23 (containing SEQ ID NO: 17). Shown are the means (±standard deviation) of 4 individual control plants and 3 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.285 ± 0.022 OSW23 transgenic seeds 0.317 ± 0.008
TABLE-US-00029 TABLE 28 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW24 (containing SEQ ID NO: 19). Shown are the means (±standard deviation) of 4 individual control plants and 6 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.267 ± 0.029 OSW24 transgenic seeds 0.307 ± 0.009
TABLE-US-00030 TABLE 29 Determination of the T2 seeds of total fatty acid content of transgenic lines of OSW26 (containing SEQ ID NO: 21). Shown are the means (±standard deviation) of 4 individual control plants and 5 individual plants per line Genotype % total fatty acids/g seed weight Col-0 empty vector control 0.259 ± 0.012 OSW26 transgenic seeds 0.287 ± 0.014
TABLE-US-00031 TABLE 30 Determination of the T2 seeds of total fatty acid content of transgenic lines of ToZ01 (containing SEQ ID NO: 47). Shown are the means (±standard deviation) of 4 individual control plants and 7 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.280 ± 0.011 ToZ01 transgenic seeds 0.298 ± 0.003
TABLE-US-00032 TABLE 31 Determination of the T2 seeds of total fatty acid content of transgenic lines of ToZ02 (containing SEQ ID NO: 49). Shown are the means (±standard deviation) of 4 individual control plants and 7 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.247 ± 0.011 ToZ02 transgenic seeds 0.278 ± 0.019
TABLE-US-00033 TABLE 32 Determination of the T2 seeds of total fatty acid content of transgenic lines of ToZ03 (containing SEQ ID NO: 51). Shown are the means (±standard deviation) of 4 individual control plants and 2 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.276 ± 0.011 ToZ03 transgenic seeds 0.312 ± 0.027
TABLE-US-00034 TABLE 33 Determination of the T2 seeds of total fatty acid content of transgenic lines of osw20 suppression (containing SEQ ID NO: X). Shown are the means (±standard deviation) of 4 individual control plants and 14 individual plants per line Genotype g total fatty acids/g seed weight Col-0 empty vector control 0.338 ± 0.022 Osw20 suppression transgenic seeds 0.379 ± 0.012
Analysis of the Impact of Recombinant Proteins on the Production of a Desired Seed Storage Compound
The effect of the genetic modification in plants on a desired seed storage compound (such as a sugar, lipid, or fatty acid) can be assessed by growing the modified plant under suitable conditions and analyzing the seeds or any other plant organ for increased production of the desired product (i.e., a lipid or a fatty acid). Such analysis techniques are well known to one skilled in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (See, for example, Ullman, 1985, Encyclopedia of Industrial Chemistry, vol. A2, pp. 89-90 and 443-613, VCH: Weinheim; Fallon, A. et al., 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993, Product recovery and purification, Biotechnology, vol. 3, Chapter III, pp. 469-714, VCH: Weinheim; Belter, P. A. et al., 1988, Bioseparations: downstream processing for biotechnology, John Wiley & Sons; Kennedy J. F. & Cabral J. M. S., 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz J. A. & Henry J. D., 1988, Biochemical separations in: Ulmann's Encyclopedia of Industrial Chemistry, Separation and purification techniques in biotechnology, vol. B3, Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow F. J. 1989).
Besides the above-mentioned methods, plant lipids are extracted from plant material as described by Cahoon et al. (1999, Proc. Natl. Acad. Sci. USA 96, 22:12935-12940) and Browse et al. (1986, Anal. Biochemistry 442:141-145). Qualitative and quantitative lipid or fatty acid analysis is described in Christie, William W., Advances in Lipid Methodology. Ayr/Scotland: Oily Press.--(Oily Press Lipid Library; Christie, William W., Gas Chromatography and Lipids. A Practical Guide--Ayr, Scotland :Oily Press, 1989 Repr. 1992.--IX, 307 S.--Oily Press Lipid Library; and Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16 (1977) Progress in the Chemistry of Fats and Other Lipids CODEN).
Unequivocal proof of the presence of fatty acid products can be obtained by the analysis of transgenic plants following standard analytical procedures: GC, GC-MS or TLC as variously described by Christie and references therein (1997 in: Advances on Lipid Methodology 4th ed.: Christie, Oily Press, Dundee, pp. 119-169; 1998). Detailed methods are described for leaves by Lemieux et al. (1990, Theor. Appl. Genet. 80:234-240) and for seeds by Focks & Benning (1998, Plant Physiol. 118:91-101).
Positional analysis of the fatty acid composition at the C-1, C-2, or C-3 positions of the glycerol backbone is determined by lipase digestion (See, e.g., Siebertz & Heinz 1977, Z. Naturforsch. 32c:193-205, and Christie, 1987, Lipid Analysis 2nd Edition, Pergamon Press, Exeter, ISBN 0-08-023791-6).
A typical way to gather information regarding the influence of increased or decreased protein activities on lipid and sugar biosynthetic pathways is for example via analyzing the carbon fluxes by labeling studies with leaves or seeds using 14C-acetate or 14C-pyruvate (See, e.g. Focks & Benning, 1998, Plant Physiol. 118:91-101; Eccleston & Ohlrogge, 1998, Plant Cell 10:613-621). The distribution of 14C into lipids and aqueous soluble components can be determined by liquid scintillation counting after the respective separation (for example on TLC plates) including standards like 14C-sucrose and 14C-malate (Eccleston & Ohlrogge, 1998, Plant Cell 10:613-621).
Material to be analyzed can be disintegrated via sonification, glass milling, liquid nitrogen and grinding, or via other applicable methods. The material has to be centrifuged after disintegration. The sediment is resuspended in distilled water, heated for 10 minutes at 100° C., cooled on ice and centrifuged again, followed by extraction in 0.5 M sulfuric acid in methanol containing 2% dimethoxypropane for 1 hour at 90° C., leading to hydrolyzed oil and lipid compounds resulting in transmethylated lipids. These fatty acid methyl esters are extracted in petrolether and finally subjected to GC analysis using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) at a temperature gradient between 170° C. and 240° C. for 20 minutes and 5 minutes at 240° C. The identity of resulting fatty acid methylesters is defined by the use of standards available from commercial sources (e.g., Sigma).
In the case of fatty acids where standards are not available, molecule identity is shown via derivatization and subsequent GC-MS analysis. For example, the localization of triple bond fatty acids is shown via GC-MS after derivatization via 4,4-Dimethoxy-oxazolin-Derivaten (Christie, Oily Press, Dundee, 1998).
A common standard method for analyzing sugars, especially starch, is published by Stitt M., Lilley R. Mc. C., Gerhardt R. and Heldt M. W. (1989, "Determination of metabolite levels in specific cells and subcellular compartments of plant leaves," Methods Enzymol. 174:518-552; for other methods, see also Hartel et al., 1998, Plant Physiol. Biochem. 36:407-417 and Focks & Benning, 1998, Plant Physiol. 118:91-101).
For the extraction of soluble sugars and starch, 50 seeds are homogenized in 500 μl of 80% (v/v) ethanol in a 1.5-ml polypropylene test tube and incubated at 70° C. for 90 minutes. Following centrifugation at 16,000 g for 5 minutes, the supernatant is transferred to a new test tube. The pellet is extracted twice with 500 μl of 80% ethanol. The solvent of the combined supernatants is evaporated at room temperature under a vacuum. The residue is dissolved in 50 μl of water, representing the soluble carbohydrate fraction. The pellet left from the ethanol extraction, which contains the insoluble carbohydrates including starch, is homogenized in 200 μl of 0.2 N KOH, and the suspension is incubated at 95° C. for 1 hour to dissolve the starch. Following the addition of 35 μl of 1 N acetic acid and centrifugation for 5 minutes at 16,000 g, the supernatant is used for starch quantification.
To quantify soluble sugars, 10 μl of the sugar extract is added to 990 μl of reaction buffer containing 100 mM imidazole, pH 6.9, 5 mM MgCl2, 2 mM NADP, 1 mM ATP, and 2 units 2 ml-1 of Glucose-6-P-dehydrogenase. For enzymatic determination of glucose, fructose, and sucrose, 4.5 units of hexokinase, 1 unit of phosphoglucoisomerase, and 2 μl of a saturated fructosidase solution are added in succession. The production of NADPH is photometrically monitored at a wavelength of 340 nm. Similarly, starch is assayed in 30 μl of the insoluble carbohydrate fraction with a kit from Boehringer Mannheim.
An example for analyzing the protein content in leaves and seeds can be found by Bradford M. M. (1976, "A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein dye binding," Anal. Biochem. 72:248-254). For quantification of total seed protein, 15-20 seeds are homogenized in 250 μl of acetone in a 1.5-ml polypropylene test tube. Following centrifugation at 16,000 g, the supernatant is discarded and the vacuum-dried pellet is resuspended in 250 μl of extraction buffer containing 50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1 mM EDTA, and 1% (w/v) SDS. Following incubation for 2 hours at 25° C., the homogenate is centrifuged at 16,000 g for 5 minutes, and 200 ml of the supernatant will be used for protein measurements. In the assay, γ-globulin is used for calibration. For protein measurements, Lowry DC protein assay (Bio-Rad) or Bradford-assay (Bio-Rad) are used.
Enzymatic assays of hexokinase and fructokinase are performed spectrophotometrically according to Renz et al. (1993, Planta 190:156-165); enzymatic assays of phosphogluco-isomerase, ATP-dependent 6-phosphofructokinase, pyrophosphate-dependent 6-phospho-fructokinase, Fructose-1,6-bisphosphate aldolase, triose phosphate isomerase, glyceral-3-P dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase are performed according to Burrell et al. (1994, Planta 194:95-101); and enzymatic assays of UDP-Glucose-pyrophosphorylase according to Zrenner et al. (1995, Plant J. 7:97-107).
Intermediates of the carbohydrate metabolism, like Glucose-1-phosphate, Glucose-6-phosphate, Fructose-6-phosphate, Phosphoenolpyruvate, Pyruvate, and ATP are measured as described in Hartel et al. (1998, Plant Physiol. Biochem. 36:407-417), and metabolites are measured as described in Jelitto et al. (1992, Planta 188:238-244).
In addition to the measurement of the final seed storage compound (i.e., lipid, starch or storage protein), it is also possible to analyze other components of the metabolic pathways utilized for the production of a desired seed storage compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound (Fiehn et al., 2000, Nature Biotech. 18:1447-1161).
For example, yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for alterations in sugar, oil, lipid, or fatty acid contents.
Similarly, plant expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soybean, rape, maize, wheat, Medicago truncatula, etc., using standard protocols. The resulting transgenic cells and/or plants derived therefrom can then be assayed for alterations in sugar, oil, lipid, or fatty acid contents.
Additionally, the sequences disclosed herein, or fragments thereof, can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke at al., 1998, Plant J. 15:39-48). The resultant knockout cells can then be evaluated for their composition and content in seed storage compounds, and the effect on the phenotype and/or genotype of the mutation. For other methods of gene inactivation include U.S. Pat. No. 6,004,804 and Puttaraju et al., 1999, Nature Biotech. 17:246-252).
Purification of the Desired Product from Transformed Organisms
An LMP can be recovered from plant material by various methods well known in the art. Organs of plants can be separated mechanically from other tissue or organs prior to isolation of the seed storage compound from the plant organ. Following homogenization of the tissue, cellular debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from cells grown in culture, then the cells are removed from the culture by low-speed centrifugation, and the supernatant fraction is retained for further purification.
The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One skilled in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey J. E. & Ollis D. F., 1986, Biochemical Engineering Fundamentals, McGraw-Hill: New York.
The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, analytical chromatography such as high performance liquid chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994, Appl. Environ. Microbiol. 60:133-140), Malakhova et al. (1996, Biotekhnologiya 11:27-32), Schmidt et al. (1998, Bioprocess Engineer 19:67-70), Ulmann's Encyclopedia of Industrial Chemistry (1996, Vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587) and Michal G. (1999, Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17).
Screening for Increased Stress Tolerance and Plant Growth
The transgenic plants are screened for their improved stress tolerance demonstrating that transgene expression confers stress tolerance. The transgenic plants are further screened for their growth rate demonstrating that transgene expression confers increased growth rates and/or increased seed yield.
Classification of the proteins was done by Blasting against the BLOCKS database (S. Henikoff & J. G. Henikoff, Genomics 19:97-107 (1994)).
Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompasses by the claims to the invention disclosed and claimed herein.
Construction of a Binary Construct for Suppression of Translation of osw20
The sequence of osw 20 (SEQ ID NO:11) was cloned using the forward and reverse primers (SEQ ID NOS:95 and 96) as described above. A construct for suppression of osw20 by mRNA interference was constructed as follows. The plasmid pGEM-Te carrying SEQ ID NO:11 (amplified with SEQ ID NOS:95 and 96) was digested with the restriction enzyme StuI, making use of the restriction site introduced by the reverse primer. After ethanol precipitation, the plasmid was digested with the restriction enzyme PstI making use of the PstI site introduced by the forward primer. The resulting liberated nucleotide was purified from an agarose gel using standard methods and digested with the restriction enzymes HindIII and NdeI simultaneously. HindIII cut the sequence in two fragments of the same size, and NdeI cleaved the rear half of the original nucleotide into two smaller fragments. The front half of the osw20 sequence (approx 530 bp, between the restriction sites of Pst I and Hind III) was purified from an agarose gel and the nucleotide overhangs filled up to blunt ends using the large fragment of the Klenow polymerase.
The plasmid pGEM-Te carrying the osw20 sequence was digested with StuI, and the blunted osw20 fragment was ligated behind the osw20 sequence. The resulting clones were screened for one with a reverse orientation of the inserted fragment behind the open reading frame of osw20. This construct was called osw20 suppression (SEQ ID NO:123). It was liberated from the pGEM-Te vector by a digestion with NotI and gel purification, and ligated into the binary vector pSUN300 under control of the seed specific USP promoter (See FIG. 1).
TABLE-US-00035 Appendix Nucleic Acid Sequence of Osw26 (SEQ ID NO: 121) ATGCAGACCGTTTCTCGGAGATTAGCTCGTGAAAATTTGAGCTCTCGCAC ATCGATTTACTCTCTCAAATCGCTTTATCCTGTTTCCGATCGCTGTTACG GTGAGTATGATCGGCGTTATGCCTCTACGCTTACCACCAAAGGTATTGGA CATCTGGTCCGCAAGGGTACTGGTGGAAGATCGTCTGTTAGTGGGATAGT TGCTACAGTATTCGGAGCTACTGGTTTCCTTGGGCGTTACTTGGTGCAAC AGCTTGCTAAAACGGGTTCACAAGTGCTAGTACCATTTAGAGGTTCCGAA GATTCGCCCCGTCATCTCAAACTGATGGGCGATTTGGGGCAGATTGTTCC CATGAAATATAATCCTAGAGATGAAAACTCAATTAAGGCAGTCATGGCCA AGGCAAATGTTGTGATTAATCTCATAGGAAGGGAATATGAAACCAGAAAT TATAGTTTTGAGGAAGTGAACCATCATATGGCTGAACAACTTGCAAAGAT TTTCCAAAGAACATGGTGGAATCATGAGATTTATACAACTGTCGTGTTTA GGTGCATCTAAATCATCTCCATCTAGGATGCTTCAAGCCAAGGCTGCTGC AGAAGAATCCATCTTACGTGAATTGCCTGAGGCCACAATACTGCGACCTG CAGTGATGGTTGGTACAGAAGATCGGATCTTGAACCCATGGGCTCAGTTC GCTAAAAAATATAACTTTCTTCCAATGATCGGGGGTGGTTCTACTAAGAT TCAGCCTGTGTATGTTGCTGATGTCGCCTCTGCAGTTGTTGCGGCATTAA GTGATGACGGTAGTAGCATGGGAAAAGTGTATGAACTTGGTGGGCCTGAT GTTTATACACTGCATCAATTGGCTGAACTTATGTATGAAACGATTCGAGA ATGGCCTCATTATGTTAACGTTCCTTTCCCTATTGCTAAGGCGATCTCAA CACCTCGAGAAGTATTTCTTAATAAAGTTCCCTTCCCGTTACCCTCACCA ATCATCTTCAATTTGGATGTGATTAATGCTCTTTCTTCAGATACTCTCGT CTCAAAAGATGCTCTGACATTCAATGATCTTGAGCTTGTGCCACATAAGG TGAAGGGATATCCTATTGAGTACCTTATCCAGTATCGCAAGGGTGGACCC AATTACGGCTCTACAGTCAGTGAAAGAGTGACTCCAGAGTCTTATCCTT GA Deduced Amino Acid Sequence of Osw26 (SEQ ID NO: 122) MQTVSRRLARENLSSRTSIYSLKSLYPVSDRCYGEYDRRYASTLTTKGIG HLVRKGTGGRSSVSGIVATVFGATGFLGRYLVQQLAKTGSQVLVPFRGSE DSPRHLKLMGDLGQIVPMKYNPRDENSIKAVMAKANVVINLIGREYETRN YSFEEVNHHMAEQLAKISKEHGGIMRFIQLSCLGASKSSPSRMLQAKAAA EESILRELPEATILRPAVMVGTEDRILNPWAQFAKKYNFLPMIGGGSTKI QPVYVADVASAVVAALSDDGSSMGKVYELGGPDVYTLHQLAELMYLTIRE WPHYVNVPFPIAKAISTPREVFLNKVPFPLPSPILFNLDVINALSSDTLV SKDALTFNDLELVPHKVKGYPIEYLIQYRKGGPNYGSTVSERVTPESYP Nucleic Acid Sequence of Osw20 Suppression Construct (SEQ ID NO: 123) ATGGAGTGTAGTTCAGTGAGTGTACTAGGAATATTACTGGTATTTCCTCT CCTTCATAACCTTGTCACCATCTCCGGGCAGAATCTTCCGGCGGTGGGTT TGTTCACTTTCGGAGATTCCAACTTCGACGCTGGAAATAAAAAGTTCCTC ACAAGTGCTCCACTTCCTCAAAACTTTTGGCCTTACGGTAAATCTCGAGA TGACCCTAAGGGCAAGTTTTCTGATGGCAAAATTGTCCCGGACTTTATTG CAAAATTCATGGGGATACCACACGATTTACCGCCGGCGCTAAAACCCGGC ACCGATGTGTCACGAGGAGCCAGCTTCGCCGTCGGGTCCGCTTCCATTCT TGGATCTCCAAAAGATTCTTTGGCTCTGAATCAACAAGTGAGGAAATTCA ATCAGATGATATCAAATTGGAAAGTGGATTACATTCAGAAATCAGTGTTT ATGATTAGCATTGGTATGGAAGATTACTACAACTTTACCAAAAACAATCC TAATGCTGAAGTTTCTGCTCAACAAGCTTTCGTTACTTCTGTCACTAACC GGTTTAAGAGTGATATCAACTTGTTGTATTCATCTGGAGCTAGTAAATTC GTCGTACACTTGCTAGCGCCATTAGGTTGTTTACCGATCGCAAGACAAGA ATTTAAAACCGGTAACAATTGTTACGAGAAACTCGATGATTTGGCCAAAC AACACAACGCTAAAATTGGACCGATTTTGAACGAAATGGCGGAAACTAAA CCGGATTTCCAATTCACCGTTTTCGATTTCTACAACGTTATTCTTCGCAG GACACAAAGAAACATGAACTACCGGTTTTCCGTGACGAATATATCGTGTT GCGGTGTTGGGACGCATTATGCATATGGTTGTGGTTTACCTAACGTGCAC TCGAAGTTATGCGAATATCAAAGATCCTACCTTTACTTCGACGCACGTCA TAACACAGAGAAAGCACAAGAAGCGTTTGCTCATCTTATCTTTGGAGCTG ACCCAAATGTTATCCAACCTATGAATGTTCGTGAGCTCATGGTGTATCCT GTTAATGAGCCTATGCGTGAGTTTTGGGAGGATCCAATGGATGAGAAGTT ATCGTTAGTCCAATACTAGAGGAGCTTGTTGAGCAGAAACTTCAGCATTA GGATTGTTTTTGGTAAAGTTGTAGTAATCTTCCATACCAATGCTAATCAT AAACACTGATTTCTGAATGTAATCCACTTTCCAATTTGATATCATCTGAT TGAATTTCCTCACTTGTTGATTCAGAGCCAAAGAATCTTTTGGAGATCCA AGAATGGAAGCGGACCCGACGGCGAAGCTGGCTCCTCGTGACACATCGGT GCCGGGTTTTAGCGCCGGCGGTAAATCGTGTGGTATCCCCATGAATTTTG CAATAAAGTCCGGGACAATTTTGCCATCAGAAAACTTGCCCTTAGGGTCA TCTCGAGATTTACCGTAAGGCCAAAAGTTTTGAGGAAGTGGAGCACTTGT GAGGAACTTTTTATTTCCAGCGTCGAAGTTGGAATCTCCGAAAGTGAACA AACCCACCGCCGGAAGATTCTGCCCGGAGATGGTGACAAGGTTATGAAGG AGAGGAAATACCAGTAATATTCCTAGTACACTCACTGAACTACACTCCAT GGC
12312976DNAArabidopsis thaliana 1atggcaacgg gggctgagaa ccaatggctt aaaggaagag tgaaggctgt tacctccgga 60gactgcttag tgatcacggc tttgagccac aacagagctg gacctccacc ggaaaagacc 120attacttttt cttctcttat ggcacctaag atggctcgca gaggaggtat agatgagcct 180tttgcatggg aaagcaaaga atttttgagg aaactttgca taggaaagga ggttgcattc 240aaagtggatt acaaggtgga agctattgct ggaagagaat ttggctctgt tttccttggc 300aacgagaatc ttgctaagct tgttgttaaa actggttggg caaaggttag ggagccaggt 360cagcagaatc aggacaaggt tagtccttac attaaagagt tgctacagct tgaagagctg 420gccaagcagg aaggatatgg tcgttggagc aaggttcctg gtgctgctga ggcatctatc 480agaaatcttc ctccttctgc cattggggat tctgctggct ttgatgccat gggcctttta 540gctgcaaaca agggcaagcc tatggaaggt attgtagagc aagtgcgtga tggaagtact 600attcgggttt atcttcttcc agagttccag tttgtgcaag tatttgttgc gggagtccag 660gctccatcaa tgggaaggcg aaccacaaat ggaagtgttg ttgagacagt tccagatgag 720ccgaatggag atgtttctgc tgagtcacga ggtcctctaa cgacagctca gagacttgct 780gcctctgcag catcgtctgt cgaggtttcc tctgatccat ttgcaactga agccaagtac 840tttaccgaac accgtgttct tagtagagat gttcgcattg ttcttgaagg cgttgacaaa 900ttcaacaatc tgattggttc agttcattat tctgatgggg aaacagttaa agacttgggg 960ttggagctcg ttgaaaatgg tcttgctaaa tttgttgaat ggagtgccaa catgatggag 1020gaggaagcta agaaaaagtt gaaagctgca gaacttcaat gcaagaaaga taaggttaaa 1080atgtgggcaa actacgttcc tccagctaca aactctaagg caattcatga ccagaacttt 1140acgggaaagg tagtggaagt ggtgagtggg gattgtctaa tagttgccga tgatgctgtt 1200ccatttggga gtccagcagc agagagacgg gtctgtcttt cgagtatcag atctccaaaa 1260atgggcaacc cacgtagaga agagaaacca gctccttatg ctcgggaagc aagagaattt 1320ctgagacaac gacttattgg caaacaggtt attgttcaaa tggaatattc aaggaaagtc 1380acccaaggag atggtcctac cacatctgga gctgctgata ggttcatgga ttttggctca 1440gtgttccttc catctgctgc caaagccgat tctgatgaag tgactgcacc acctgctgca 1500gcaattgctg gcagtcagcc ggttggtgtg aatattgctg agctcgttct tgtccgtggt 1560tttggaaatg tggttagaca tagagatttt gaagagcgat caaaccatta tgatgctctt 1620ctggctgctg aagctcgtgc tctggctgga aagaaaggaa tccattctgc aaaagaatct 1680ccagccatgc acatcacaga cctaactgtg tcggcagcta agaaagctaa agatttcctg 1740ccatccctgc aaagaatcag gagaataccc gctgttgtgg aatatgtcct cagcggacat 1800cggtttaagc tttatatccc aaagataaca tgtagcatag ccttttcatt ctctggtgtc 1860agatgtcctg gccgtggcga accttattca gaagaagcta tctctgtaat gagacgtagg 1920atcatgcaga gagatgttga gattgaagtt gaaaccgtgg atagaaccgg tactttcttg 1980ggatccatgt gggaatcgag gacaaacgtg gctacagttc tgcttgaagc tggcttagca 2040aaaatgcaga ctagctttgg tgcagacagg atcgccgaag cacatcttct tgaacaggca 2100gagagatctg ctaaaaacca gaaactgaag atttgggaaa actatgttga aggagaagaa 2160gtctcaaacg gaaatactaa caccgtagaa accaggcaaa aggagacctt aaaggttgtt 2220gttacagaag tacttggagg tggtcggttc tatgttcaat ctgctggaga tcagaaaata 2280gcttcgattc agaaccagct tgcatcattg agtattaaag acgctcccat tatcggatcc 2340tttaatccaa agagaggtga catcgtcctt gcacagttta gccttgataa ctcctggaac 2400cgtgcaatga ttgtgacagc accccgagca gcggttcaat ccccagatga aaaattcgaa 2460gtgttctaca tcgattatgg aaaccaagag acagttccat acagcgcaat cagaccaata 2520gacccttcgg tatctgcagc accagggctc gctcagctct gcagacttgc ctacataaag 2580gttccaagct tggaagacga ctttggtcct gaagcgggag agtatttgca tactgtaact 2640ctgggtagtg gtaaagagtt caaagcagtg atagaagaaa gagacacatc tggaggcaaa 2700gtcaaagggc aaggcactgg aactgaattc gttgtcactc tcattgctgt tgatgatgag 2760atctctgtaa atgctgcaat gcttcaggaa ggaatagcga gaatggagaa acgtcagaaa 2820tgggggcaca aaggcaaaca agctgctctt gatgctttag agaagttcca agaggaagct 2880cgcaagtcga gaattggaat ctggcagtac ggtgacattg agtccgatga tgaggacact 2940ggtccggcca gaaagcctgc tggtggtcgc cggtaa 29762991PRTArabidopsis thaliana 2Met Ala Thr Gly Ala Glu Asn Gln Trp Leu Lys Gly Arg Val Lys Ala1 5 10 15Val Thr Ser Gly Asp Cys Leu Val Ile Thr Ala Leu Ser His Asn Arg 20 25 30Ala Gly Pro Pro Pro Glu Lys Thr Ile Thr Phe Ser Ser Leu Met Ala 35 40 45Pro Lys Met Ala Arg Arg Gly Gly Ile Asp Glu Pro Phe Ala Trp Glu 50 55 60Ser Lys Glu Phe Leu Arg Lys Leu Cys Ile Gly Lys Glu Val Ala Phe65 70 75 80Lys Val Asp Tyr Lys Val Glu Ala Ile Ala Gly Arg Glu Phe Gly Ser 85 90 95Val Phe Leu Gly Asn Glu Asn Leu Ala Lys Leu Val Val Lys Thr Gly 100 105 110Trp Ala Lys Val Arg Glu Pro Gly Gln Gln Asn Gln Asp Lys Val Ser 115 120 125Pro Tyr Ile Lys Glu Leu Leu Gln Leu Glu Glu Leu Ala Lys Gln Glu 130 135 140Gly Tyr Gly Arg Trp Ser Lys Val Pro Gly Ala Ala Glu Ala Ser Ile145 150 155 160Arg Asn Leu Pro Pro Ser Ala Ile Gly Asp Ser Ala Gly Phe Asp Ala 165 170 175Met Gly Leu Leu Ala Ala Asn Lys Gly Lys Pro Met Glu Gly Ile Val 180 185 190Glu Gln Val Arg Asp Gly Ser Thr Ile Arg Val Tyr Leu Leu Pro Glu 195 200 205Phe Gln Phe Val Gln Val Phe Val Ala Gly Val Gln Ala Pro Ser Met 210 215 220Gly Arg Arg Thr Thr Asn Gly Ser Val Val Glu Thr Val Pro Asp Glu225 230 235 240Pro Asn Gly Asp Val Ser Ala Glu Ser Arg Gly Pro Leu Thr Thr Ala 245 250 255Gln Arg Leu Ala Ala Ser Ala Ala Ser Ser Val Glu Val Ser Ser Asp 260 265 270Pro Phe Ala Thr Glu Ala Lys Tyr Phe Thr Glu His Arg Val Leu Ser 275 280 285Arg Asp Val Arg Ile Val Leu Glu Gly Val Asp Lys Phe Asn Asn Leu 290 295 300Ile Gly Ser Val His Tyr Ser Asp Gly Glu Thr Val Lys Asp Leu Gly305 310 315 320Leu Glu Leu Val Glu Asn Gly Leu Ala Lys Phe Val Glu Trp Ser Ala 325 330 335Asn Met Met Glu Glu Glu Ala Lys Lys Lys Leu Lys Ala Ala Glu Leu 340 345 350Gln Cys Lys Lys Asp Lys Val Lys Met Trp Ala Asn Tyr Val Pro Pro 355 360 365Ala Thr Asn Ser Lys Ala Ile His Asp Gln Asn Phe Thr Gly Lys Val 370 375 380Val Glu Val Val Ser Gly Asp Cys Leu Ile Val Ala Asp Asp Ala Val385 390 395 400Pro Phe Gly Ser Pro Ala Ala Glu Arg Arg Val Cys Leu Ser Ser Ile 405 410 415Arg Ser Pro Lys Met Gly Asn Pro Arg Arg Glu Glu Lys Pro Ala Pro 420 425 430Tyr Ala Arg Glu Ala Arg Glu Phe Leu Arg Gln Arg Leu Ile Gly Lys 435 440 445Gln Val Ile Val Gln Met Glu Tyr Ser Arg Lys Val Thr Gln Gly Asp 450 455 460Gly Pro Thr Thr Ser Gly Ala Ala Asp Arg Phe Met Asp Phe Gly Ser465 470 475 480Val Phe Leu Pro Ser Ala Ala Lys Ala Asp Ser Asp Glu Val Thr Ala 485 490 495Pro Pro Ala Ala Ala Ile Ala Gly Ser Gln Pro Val Gly Val Asn Ile 500 505 510Ala Glu Leu Val Leu Val Arg Gly Phe Gly Asn Val Val Arg His Arg 515 520 525Asp Phe Glu Glu Arg Ser Asn His Tyr Asp Ala Leu Leu Ala Ala Glu 530 535 540Ala Arg Ala Leu Ala Gly Lys Lys Gly Ile His Ser Ala Lys Glu Ser545 550 555 560Pro Ala Met His Ile Thr Asp Leu Thr Val Ser Ala Ala Lys Lys Ala 565 570 575Lys Asp Phe Leu Pro Ser Leu Gln Arg Ile Arg Arg Ile Pro Ala Val 580 585 590Val Glu Tyr Val Leu Ser Gly His Arg Phe Lys Leu Tyr Ile Pro Lys 595 600 605Ile Thr Cys Ser Ile Ala Phe Ser Phe Ser Gly Val Arg Cys Pro Gly 610 615 620Arg Gly Glu Pro Tyr Ser Glu Glu Ala Ile Ser Val Met Arg Arg Arg625 630 635 640Ile Met Gln Arg Asp Val Glu Ile Glu Val Glu Thr Val Asp Arg Thr 645 650 655Gly Thr Phe Leu Gly Ser Met Trp Glu Ser Arg Thr Asn Val Ala Thr 660 665 670Val Leu Leu Glu Ala Gly Leu Ala Lys Met Gln Thr Ser Phe Gly Ala 675 680 685Asp Arg Ile Ala Glu Ala His Leu Leu Glu Gln Ala Glu Arg Ser Ala 690 695 700Lys Asn Gln Lys Leu Lys Ile Trp Glu Asn Tyr Val Glu Gly Glu Glu705 710 715 720Val Ser Asn Gly Asn Thr Asn Thr Val Glu Thr Arg Gln Lys Glu Thr 725 730 735Leu Lys Val Val Val Thr Glu Val Leu Gly Gly Gly Arg Phe Tyr Val 740 745 750Gln Ser Ala Gly Asp Gln Lys Ile Ala Ser Ile Gln Asn Gln Leu Ala 755 760 765Ser Leu Ser Ile Lys Asp Ala Pro Ile Ile Gly Ser Phe Asn Pro Lys 770 775 780Arg Gly Asp Ile Val Leu Ala Gln Phe Ser Leu Asp Asn Ser Trp Asn785 790 795 800Arg Ala Met Ile Val Thr Ala Pro Arg Ala Ala Val Gln Ser Pro Asp 805 810 815Glu Lys Phe Glu Val Phe Tyr Ile Asp Tyr Gly Asn Gln Glu Thr Val 820 825 830Pro Tyr Ser Ala Ile Arg Pro Ile Asp Pro Ser Val Ser Ala Ala Pro 835 840 845Gly Leu Ala Gln Leu Cys Arg Leu Ala Tyr Ile Lys Val Pro Ser Leu 850 855 860Glu Asp Asp Phe Gly Pro Glu Ala Gly Glu Tyr Leu His Thr Val Thr865 870 875 880Leu Gly Ser Gly Lys Glu Phe Lys Ala Val Ile Glu Glu Arg Asp Thr 885 890 895Ser Gly Gly Lys Val Lys Gly Gln Gly Thr Gly Thr Glu Phe Val Val 900 905 910Thr Leu Ile Ala Val Asp Asp Glu Ile Ser Val Asn Ala Ala Met Leu 915 920 925Gln Glu Gly Ile Ala Arg Met Glu Lys Arg Gln Lys Trp Gly His Lys 930 935 940Gly Lys Gln Ala Ala Leu Asp Ala Leu Glu Lys Phe Gln Glu Glu Ala945 950 955 960Arg Lys Ser Arg Ile Gly Ile Trp Gln Tyr Gly Asp Ile Glu Ser Asp 965 970 975Asp Glu Asp Thr Gly Pro Ala Arg Lys Pro Ala Gly Gly Arg Arg 980 985 99031014DNAArabidopsis thaliana 3atggcaacca tggctaggtc gtttctgcag gcgatatcga aggatgaggc ggtggctcct 60ccgcttagag ttgttcagat cgaaggactg gctgtactaa agataatcaa acactgcaag 120gagttttcac cgacccttgt cactggacag cttcttggac ttgatgttgg tagcgtcctc 180gaagttacca attgttttcc tttcccggtc agggatgatg atgaagaaat tgaagctgat 240ggtgctaatt atcagcttga gatgatgaga tgtctgaggg aggttaatgt tgacaacaac 300actgttggct ggtatcaatc tacagttctt ggttcgtatc agactgtgga actgattgag 360accttcatga attaccagga gaatatcaag aggtgtgtgt gcatcatata tgatccctct 420aaagctgatc taggcgtctt agctttgaag gctttgaagc tttcagattc ctttatggag 480ttgtaccgag gtggaaactt tactggcgag aagttgagag agaaaaattt ctcctggatg 540gatatttttg aggaaatacc tatcaaggtt tcaaattctg cccttgtcag tgcctttatg 600accgaactgg agactgatac acctgtctca cagggcgatt atgatcgtct acactcatca 660accactcctt tccttgagaa caatatggag tttttgatta aatgcatgga tgatttatct 720atggaacagc aaaagttcca gtattactac cggaacctgt ctcgtcagca agcacaacag 780caagcctggc tccagaagag aagaacggag aacatggctc gtaaatcagc tggagaggag 840cctttaccag aagaggatcc ttcaaaccca atctttaagg cgatccctga accatctagg 900ctagagagtt tcctcatcac aaaccaagtc tcaaacttct gtggccaaat caatggagtg 960gctggccaga acttcagcag gctttacctg accaaagcat tgcacgacaa ctga 10144337PRTArabidopsis thaliana 4Met Ala Thr Met Ala Arg Ser Phe Leu Gln Ala Ile Ser Lys Asp Glu1 5 10 15Ala Val Ala Pro Pro Leu Arg Val Val Gln Ile Glu Gly Leu Ala Val 20 25 30Leu Lys Ile Ile Lys His Cys Lys Glu Phe Ser Pro Thr Leu Val Thr 35 40 45Gly Gln Leu Leu Gly Leu Asp Val Gly Ser Val Leu Glu Val Thr Asn 50 55 60Cys Phe Pro Phe Pro Val Arg Asp Asp Asp Glu Glu Ile Glu Ala Asp65 70 75 80Gly Ala Asn Tyr Gln Leu Glu Met Met Arg Cys Leu Arg Glu Val Asn 85 90 95Val Asp Asn Asn Thr Val Gly Trp Tyr Gln Ser Thr Val Leu Gly Ser 100 105 110Tyr Gln Thr Val Glu Leu Ile Glu Thr Phe Met Asn Tyr Gln Glu Asn 115 120 125Ile Lys Arg Cys Val Cys Ile Ile Tyr Asp Pro Ser Lys Ala Asp Leu 130 135 140Gly Val Leu Ala Leu Lys Ala Leu Lys Leu Ser Asp Ser Phe Met Glu145 150 155 160Leu Tyr Arg Gly Gly Asn Phe Thr Gly Glu Lys Leu Arg Glu Lys Asn 165 170 175Phe Ser Trp Met Asp Ile Phe Glu Glu Ile Pro Ile Lys Val Ser Asn 180 185 190Ser Ala Leu Val Ser Ala Phe Met Thr Glu Leu Glu Thr Asp Thr Pro 195 200 205Val Ser Gln Gly Asp Tyr Asp Arg Leu His Ser Ser Thr Thr Pro Phe 210 215 220Leu Glu Asn Asn Met Glu Phe Leu Ile Lys Cys Met Asp Asp Leu Ser225 230 235 240Met Glu Gln Gln Lys Phe Gln Tyr Tyr Tyr Arg Asn Leu Ser Arg Gln 245 250 255Gln Ala Gln Gln Gln Ala Trp Leu Gln Lys Arg Arg Thr Glu Asn Met 260 265 270Ala Arg Lys Ser Ala Gly Glu Glu Pro Leu Pro Glu Glu Asp Pro Ser 275 280 285Asn Pro Ile Phe Lys Ala Ile Pro Glu Pro Ser Arg Leu Glu Ser Phe 290 295 300Leu Ile Thr Asn Gln Val Ser Asn Phe Cys Gly Gln Ile Asn Gly Val305 310 315 320Ala Gly Gln Asn Phe Ser Arg Leu Tyr Leu Thr Lys Ala Leu His Asp 325 330 335Asn52496DNAArabidopsis thaliana 5atggcgcaac ccctcgtgaa gaaagacgat gatcacgacg atgagttgga gtattctcca 60ttcatgggaa ttgagaaagg agcggttctt caagaggcta gagtctttaa tgaccctcag 120gttgatccta gacgatgctc ccaggtcatt acgaagcttc tttatttgct taaccaaggg 180gagtcattca ccaaggttga agcaacggaa gttttctttt cagttacaaa gctttttcaa 240tcaaaagaca cgggtttgag gagaatggtc tacttgatca ttaaggagtt atctccatca 300tctgatgagg ttatcatcgt aacaagctct ctgatgaagg atatgaatag taaaattgat 360atgtatcgag caaatgctat ccgtgtcctc tgccggataa tagacggaac ccttctcact 420cagattgagc gatacttgaa acaagccatt gtggataaga atcccgttgt ttcaagtgca 480gctttagtca gtgggcttca cttgctcaag acaaacccag aaattgttaa aagatggagc 540aatgaagttc aagaaggtat tcaatccaga tcagcccttg ttcagttcca tgccctagct 600ttgctccatc agatacgcca aaatgatcgc ttggctgtta gcaaattggt tggtagcttg 660accaggggat ctgtccgctc tcccttggct cagtgtcttt tgatacaacg tccgttctat 720gaatttttgg agagttgcct gcgccataag gcagaaatgg tgatccttga ggctgccagg 780gcaattactg agcttgatgg tgtgacaagc cgagaactga ctccagcaat cactgttctt 840cagctctttt tgagttcccc cagaccagtg ttgagatttg ccgctgtccg gactctgaac 900aaggttgcaa tgactcatcc tatggctgtc accaactgca acattgatat ggagagttta 960atctctgatc aaaatagaag cattgctaca ctcgcaataa ccacactatt gaaaacaggg 1020aacgaatcaa gtgtagaacg tttgatgaag cagataacta attttatgtc agatattgct 1080gatgagttca aaattgtggt cgtggacgca ataagatcgt tgtgtgtgaa attcccactg 1140aaatacagat ccttgatgac cttcttaagc aacattctta gggaagaagg tggatttgag 1200tataaaagag caatagtaga ttctattgtg accattatca gagatattcc ggatgcaaag 1260gaaagtggac tgcttcatct atgtgaattc attgaagatt gtgaattcac atatctttca 1320acacagatcc ttcattttct gggaattgag gggcctaaca cctcagatcc aagcaagtat 1380atacgatata tatataatcg tgtgcatcta gaaaacgcca ctgtccgggc tgctgctgtt 1440tccacacttg caaagtttgg gtttatggtt gaatccttga agccccggat tactgttcta 1500ttgaagcgtt gcatctatga cagtgatgat gaggtccgtg atagggcaac actatatttg 1560agtgagccct ctgaagaagc ttttgatatc aactccgtac ctaaggaagt taaatctcag 1620ccccttgcag agaagaaagc ccagggtaaa aagcccactg gtcttggtgc accaccagct 1680gcacctgctt ctggttttga tggctatgaa agacttctct catccattcc agagtttgcc 1740gcctttggaa aacttttcaa gtcttcttta cctgtggagc taactgaagc agaaacagaa 1800tacgctgtca atgttgttaa gcatatcttt gacagtcatg tggtgtttca gtacaactgc 1860actaacacaa taccagagca gttgttggag agggtactga acattgaagc tgaggaattc 1920agtgaagtaa cttcaaaggc cctaaactca cttccttacg attcacccgg tcaagccttt 1980gtggtttttg agaagccagc tggggtccct gctgttggaa agttctccaa cacattgact 2040ttcgttgtta aggaggtaca tgttgaccca agcacaggtg aagcagaaga tgatggagta 2100gaagatgagt accagctaga ggatcttgag gttgtagctg gagattacat ggtgaaagtg 2160ggtgtctcca atttcaggaa tgcgtgggaa agcatggatg aagaagatga gcgtgtagac 2220gaatatggcc ttggccaaag agagagtttg ggagaagctg taaaggctgt catggatctt 2280cttggcatgc agacttgtga ggggacggag acaattccgc tcaatgcaag gtcacacacg 2340tgtctattgt caggtgtgta cataggcaac gtgaaagtgt tagtgagggc acagtttgga 2400atggacagct caaaggacat tgcaatgaag ctgacagtta gagctgaaga cgtttctgtc 2460gccgaggcca ttcacgagat tgttgccagc ggctaa 24966886PRTArabidopsis thaliana 6Met Ala Gln Pro Leu Val Lys Lys Asp Asp Asp His Asp Asp Glu Leu1 5 10 15Glu Tyr Ser Pro Phe Met Gly Ile Glu Lys Gly Ala Val Leu Gln Glu 20 25 30Ala Arg Val Phe Asn Asp Pro Gln Val Asp Pro Arg Arg Cys Ser Gln 35
40 45Val Ile Thr Lys Leu Leu Tyr Leu Leu Asn Gln Gly Glu Ser Phe Thr 50 55 60Lys Val Glu Ala Thr Glu Val Phe Phe Ser Val Thr Lys Leu Phe Gln65 70 75 80Ser Lys Asp Thr Gly Leu Arg Arg Met Val Tyr Leu Ile Ile Lys Glu 85 90 95Leu Ser Pro Ser Ser Asp Glu Val Ile Ile Val Thr Ser Ser Leu Met 100 105 110Lys Asp Met Asn Ser Lys Ile Asp Met Tyr Arg Ala Asn Ala Ile Arg 115 120 125Val Leu Cys Arg Ile Ile Asp Gly Thr Leu Leu Thr Gln Ile Glu Arg 130 135 140Tyr Leu Lys Gln Ala Ile Val Asp Lys Asn Pro Val Val Ser Ser Ala145 150 155 160Ala Leu Val Ser Gly Leu His Leu Leu Lys Thr Asn Pro Glu Ile Val 165 170 175Lys Arg Trp Ser Asn Glu Val Gln Glu Gly Ile Gln Ser Arg Ser Ala 180 185 190Leu Val Gln Phe His Ala Leu Ala Leu Leu His Gln Ile Arg Gln Asn 195 200 205Asp Arg Leu Ala Val Ser Lys Leu Val Gly Ser Leu Thr Arg Gly Ser 210 215 220Val Arg Ser Pro Leu Ala Gln Cys Leu Leu Ile Arg Tyr Thr Ser Gln225 230 235 240Val Ile Arg Asp Met Ala Asn His Gly Gln Ser Gly Glu Arg Pro Phe 245 250 255Tyr Glu Phe Leu Glu Ser Cys Leu Arg His Lys Ala Glu Met Val Ile 260 265 270Leu Glu Ala Ala Arg Ala Ile Thr Glu Leu Asp Gly Val Thr Ser Arg 275 280 285Glu Leu Thr Pro Ala Ile Thr Val Leu Gln Leu Phe Leu Ser Ser Pro 290 295 300Arg Pro Val Leu Arg Phe Ala Ala Val Arg Thr Leu Asn Lys Val Ala305 310 315 320Met Thr His Pro Met Ala Val Thr Asn Cys Asn Ile Asp Met Glu Ser 325 330 335Leu Ile Ser Asp Gln Asn Arg Ser Ile Ala Thr Leu Ala Ile Thr Thr 340 345 350Leu Leu Lys Thr Gly Asn Glu Ser Ser Val Glu Arg Leu Met Lys Gln 355 360 365Ile Thr Asn Phe Met Ser Asp Ile Ala Asp Glu Phe Lys Ile Val Val 370 375 380Val Asp Ala Ile Arg Ser Leu Cys Val Lys Phe Pro Leu Lys Tyr Arg385 390 395 400Ser Leu Met Thr Phe Leu Ser Asn Ile Leu Arg Glu Glu Gly Gly Phe 405 410 415Glu Tyr Lys Arg Ala Ile Val Asp Ser Ile Val Thr Ile Ile Arg Asp 420 425 430Ile Pro Asp Ala Lys Glu Ser Gly Leu Leu His Leu Cys Glu Phe Ile 435 440 445Glu Asp Cys Glu Phe Thr Tyr Leu Ser Thr Gln Ile Leu His Phe Leu 450 455 460Gly Ile Glu Gly Pro Asn Thr Ser Asp Pro Ser Lys Tyr Ile Arg Tyr465 470 475 480Ile Tyr Asn Arg Val His Leu Glu Asn Ala Thr Val Arg Ala Ala Ala 485 490 495Val Ser Thr Leu Ala Lys Phe Gly Phe Met Val Glu Ser Leu Lys Pro 500 505 510Arg Ile Thr Val Leu Leu Lys Arg Cys Ile Tyr Asp Ser Asp Asp Glu 515 520 525Val Arg Asp Arg Ala Thr Leu Tyr Leu Ser Val Leu Gly Gly Asp Gly 530 535 540Thr Val Asp Thr Asp Lys Glu Ser Lys Asp Phe Leu Phe Gly Ser Leu545 550 555 560Glu Val Pro Leu Val Asn Met Glu Thr Ser Leu Lys Asn Tyr Glu Pro 565 570 575Ser Glu Glu Ala Phe Asp Ile Asn Ser Val Pro Lys Glu Val Lys Ser 580 585 590Gln Pro Leu Ala Glu Lys Lys Ala Gln Gly Lys Lys Pro Thr Gly Leu 595 600 605Gly Ala Pro Pro Ala Ala Pro Ala Ser Gly Phe Asp Gly Tyr Glu Arg 610 615 620Leu Leu Ser Ser Ile Pro Glu Phe Ala Ala Phe Gly Lys Leu Phe Lys625 630 635 640Ser Ser Leu Pro Val Glu Leu Thr Glu Ala Glu Thr Glu Tyr Ala Val 645 650 655Asn Val Val Lys His Ile Phe Asp Ser His Val Val Phe Gln Tyr Asn 660 665 670Cys Thr Asn Thr Ile Pro Glu Gln Leu Leu Glu Arg Val Asn Val Ile 675 680 685Val Asp Ala Ser Glu Ala Glu Glu Phe Ser Glu Val Thr Ser Lys Ala 690 695 700Leu Asn Ser Leu Pro Tyr Asp Ser Pro Gly Gln Ala Phe Val Val Phe705 710 715 720Glu Lys Pro Ala Gly Val Pro Ala Val Gly Lys Phe Ser Asn Thr Leu 725 730 735Thr Phe Val Val Lys Glu Val Asp Pro Ser Thr Gly Glu Ala Glu Asp 740 745 750Asp Gly Val Glu Asp Glu Tyr Gln Leu Glu Asp Leu Glu Val Val Ala 755 760 765Gly Asp Tyr Met Val Lys Val Gly Val Ser Asn Phe Arg Asn Ala Trp 770 775 780Glu Ser Met Asp Glu Glu Asp Glu Arg Val Asp Glu Tyr Gly Leu Gly785 790 795 800Gln Arg Glu Ser Leu Gly Glu Ala Val Lys Ala Val Met Asp Leu Leu 805 810 815Gly Met Gln Thr Cys Glu Gly Thr Glu Thr Ile Pro Leu Asn Ala Arg 820 825 830Ser His Thr Cys Leu Leu Ser Gly Val Tyr Ile Gly Asn Val Lys Val 835 840 845Leu Val Arg Ala Gln Phe Gly Met Asp Ser Ser Lys Asp Ile Ala Met 850 855 860Lys Leu Thr Val Arg Ala Glu Asp Val Ser Val Ala Glu Ala Ile His865 870 875 880Glu Ile Val Ala Ser Gly 88571131DNAArabidopsis thaliana 7atgccgatta gccggagagt tctgacgccg atcaccgccg ctccggttat cttagccgtc 60ctttgcttct tcttttggtc atcaatcatc gggccggaca atttaaaggg cacgaaacac 120gtccttcaag atgctaagac cattcctctt cccgtcgatg gaccagagag cctagagttc 180gatccacaag gtgaaggccc ttacgttggc gtcaccgacg gtcgcattct caaatggcgc 240ggtgaagaac tcggctgggt cgatttcgcc tacacttctc ctcacagaga taactgttcg 300agtcatgagg tagtaccaag ttgtgggaga ccattgggac ttagcttcga gaggaaaaca 360ggagatttgt acatatgtga tggttacttt ggggtcatga aggtcggacc agagggaggc 420ctgggcgagt tagttgttga tgaagccgaa ggtcgtaaag ttatgtttgc caaccaaggg 480gatatagacg aagaggaaga tattttctac ttcaatgata gcagcgatac ataccatttc 540agggacgtat tttacgtgtc tttgtccggg acaaaggttg gaagagtaat tagatacgat 600atgaagaaga aagaggccaa agttattatg gacaaacttc gtttaccaaa tggtctagct 660ctaagcaaaa acggttcgtt tgtagtcaca tgcgagagta gtacgaacat ttgccataga 720atatgggtca aaggtcccaa atccggaacc aacgaggttt tcgcaacgct ccctggttcc 780ccggacaata tccggcgtac gccaacaggc gatttctggg tcgcattaca ttgcaaaaag 840aatttgttca cacgtgcagt tttgattcac acttgggtcg gaaggttttt catgaacacg 900atgaagatgg agactgtaat tcatttcatg aacggaggaa aacctcatgg aattgtcgtg 960aaactctctg gagagacagg agagattctt gagatacttg aggacagtga agggaagacg 1020gtgaaatatg ttagtgaggc ttatgagaca aaagatggaa agttatggat cggatctgtg 1080tattggccgg ccgtttgggt tcttgataca tcggtttatg attccatatg a 11318376PRTArabidopsis thaliana 8Met Pro Ile Ser Arg Arg Val Leu Thr Pro Ile Thr Ala Ala Pro Val1 5 10 15Ile Leu Ala Val Leu Cys Phe Phe Phe Trp Ser Ser Ile Ile Gly Pro 20 25 30Asp Asn Leu Lys Gly Thr Lys His Val Leu Gln Asp Ala Lys Thr Ile 35 40 45Pro Leu Pro Val Asp Gly Pro Glu Ser Leu Glu Phe Asp Pro Gln Gly 50 55 60Glu Gly Pro Tyr Val Gly Val Thr Asp Gly Arg Ile Leu Lys Trp Arg65 70 75 80Gly Glu Glu Leu Gly Trp Val Asp Phe Ala Tyr Thr Ser Pro His Arg 85 90 95Asp Asn Cys Ser Ser His Glu Val Val Pro Ser Cys Gly Arg Pro Leu 100 105 110Gly Leu Ser Phe Glu Arg Lys Thr Gly Asp Leu Tyr Ile Cys Asp Gly 115 120 125Tyr Phe Gly Val Met Lys Val Gly Pro Glu Gly Gly Leu Ala Glu Leu 130 135 140Val Val Asp Glu Ala Glu Gly Arg Lys Val Met Phe Ala Asn Gln Gly145 150 155 160Asp Ile Asp Glu Glu Glu Asp Ile Phe Tyr Phe Asn Asp Ser Ser Asp 165 170 175Thr Tyr His Phe Arg Asp Val Phe Tyr Val Ser Leu Ser Gly Thr Lys 180 185 190Val Gly Arg Val Ile Arg Tyr Asp Met Lys Lys Lys Glu Ala Lys Val 195 200 205Ile Met Asp Lys Leu Arg Leu Pro Asn Gly Leu Ala Leu Ser Lys Asn 210 215 220Gly Ser Phe Val Val Thr Cys Glu Ser Ser Thr Asn Ile Cys His Arg225 230 235 240Ile Trp Val Lys Gly Pro Lys Ser Gly Thr Asn Glu Val Phe Ala Thr 245 250 255Leu Pro Gly Ser Pro Asp Asn Ile Arg Arg Thr Pro Thr Gly Asp Phe 260 265 270Trp Val Ala Leu His Cys Lys Lys Asn Leu Phe Thr Arg Ala Val Leu 275 280 285Ile His Thr Trp Val Gly Arg Phe Phe Met Asn Thr Met Lys Met Glu 290 295 300Thr Val Ile His Phe Met Asn Gly Gly Lys Pro His Gly Ile Val Val305 310 315 320Lys Leu Ser Gly Glu Thr Gly Glu Ile Leu Glu Ile Leu Glu Asp Ser 325 330 335Glu Gly Lys Thr Val Lys Tyr Val Ser Glu Ala Tyr Glu Thr Lys Asp 340 345 350Gly Lys Leu Trp Ile Gly Ser Val Tyr Trp Pro Ala Val Trp Val Leu 355 360 365Asp Thr Ser Val Tyr Asp Ser Ile 370 37591113DNAArabidopsis thaliana 9atgcccatta atcagaaaat tccgacttgg tttgccgttc cggctgtttt tgctgtcttg 60tccgtaatct cgtatcaaac cttaattgtg ccggaaaatt tagagggcgc caaaaatgta 120ttgacaatgg ctaagaccat accaattcct gtcgctggac cggagagcat tgagtttgac 180ccaaaaggag aaggtcctta tgctgcggtc gtggacggcc gtattctcaa gtggcgcggc 240gatgatctcg gctgggttga ttttgcctac acatctcctc acagagggaa ctgttcaaaa 300actgaagtag tgcctacttg tggaaggcca ttaggactta ctttcgagaa gaaaacggga 360gatttgtaca tatgtgatgg ttacttgggg ctcatgaaag ttggaccaga gggaggcttg 420gccgagttaa ttgttgatga agccgaaggt cgtaaagtta tgtttgctaa ccaaggggat 480atagacgaag aggaagatgt cttttacttc aatgatagta gtgacaagta tcatttcagg 540gacgtatttt tcgtggctgt cagtggggag cggtcgggaa gagtgatcag atacgataag 600aagacgaaag aagccaaagt tatcatggac aatctcgttt gtaacaacgg tttggctcta 660aacaaagacc ggtcttttct aattacatgc gagtccggca caagtcttgt ccaccgatac 720tggattaaag gtcccaaagc cgggactcgt gatatctttg cgaaggtccc aggttaccct 780gacaacatcc ggctgacatc aactggagac ttttggattg gtttacactg caagaaaaat 840ctgataggga gattgattgt gaagtacaaa tggcttggga aattggtaga aaagacaatg 900aaactggagt acgtgattgc ttttattaac ggatttaaac cgcacggagt cgccgtgaaa 960atctccggcg agacgggaga ggtacttgag ttacttgagg acaaagaagg aaagacaatg 1020aagtatgtaa gcgaggctta tgaaagagat gatggaaagt tgtggtttgg gtctgtttac 1080tggccagccg tttgggttct tgatcgcaaa tga 111310370PRTArabidopsis thaliana 10Met Pro Ile Asn Gln Lys Ile Pro Thr Trp Phe Ala Val Pro Ala Val1 5 10 15Phe Ala Val Leu Ser Val Ile Ser Tyr Gln Thr Leu Ile Val Pro Glu 20 25 30Asn Leu Glu Gly Ala Lys Asn Val Leu Thr Met Ala Lys Thr Ile Pro 35 40 45Ile Pro Val Ala Gly Pro Glu Ser Ile Glu Phe Asp Pro Lys Gly Glu 50 55 60Gly Pro Tyr Ala Ala Val Val Asp Gly Arg Ile Leu Lys Trp Arg Gly65 70 75 80Asp Asp Leu Gly Trp Val Asp Phe Ala Tyr Thr Ser Pro His Arg Gly 85 90 95Asn Cys Ser Lys Thr Glu Val Val Pro Thr Cys Gly Arg Pro Leu Gly 100 105 110Leu Thr Phe Glu Lys Lys Thr Gly Asp Leu Tyr Ile Cys Asp Gly Tyr 115 120 125Leu Gly Leu Met Lys Val Gly Pro Glu Gly Gly Leu Ala Glu Leu Ile 130 135 140Val Asp Glu Ala Glu Gly Arg Lys Val Met Phe Ala Asn Gln Gly Asp145 150 155 160Ile Asp Glu Glu Glu Asp Val Phe Tyr Phe Asn Asp Ser Ser Asp Lys 165 170 175Tyr His Phe Arg Asp Val Phe Phe Val Ala Val Ser Gly Glu Arg Ser 180 185 190Gly Arg Val Ile Arg Tyr Asp Lys Lys Thr Lys Glu Ala Lys Val Ile 195 200 205Met Asp Asn Leu Val Cys Asn Asn Gly Leu Ala Leu Asn Lys Asp Arg 210 215 220Ser Phe Leu Ile Thr Cys Glu Ser Gly Thr Ser Leu Val His Arg Tyr225 230 235 240Trp Ile Lys Gly Pro Lys Ala Gly Thr Arg Asp Ile Phe Ala Lys Val 245 250 255Pro Gly Tyr Pro Asp Asn Ile Arg Leu Thr Ser Thr Gly Asp Phe Trp 260 265 270Ile Gly Leu His Cys Lys Lys Asn Leu Ile Gly Arg Leu Ile Val Lys 275 280 285Tyr Lys Trp Leu Gly Lys Leu Val Glu Lys Thr Met Lys Leu Glu Tyr 290 295 300Val Ile Ala Phe Ile Asn Gly Phe Lys Pro His Gly Val Ala Val Lys305 310 315 320Ile Ser Gly Glu Thr Gly Glu Val Leu Glu Leu Leu Glu Asp Lys Glu 325 330 335Gly Lys Thr Met Lys Tyr Val Ser Glu Ala Tyr Glu Arg Asp Asp Gly 340 345 350Lys Leu Trp Phe Gly Ser Val Tyr Trp Pro Ala Val Trp Val Leu Asp 355 360 365Arg Lys 370111119DNAArabidopsis thaliana 11atggagtgta gttcagtgag tgtactagga atattactgg tatttcctct ccttcataac 60cttgtcacca tctccgggca gaatcttccg gcggtgggtt tgttcacttt cggagattcc 120aacttcgacg ctggaaataa aaagttcctc acaagtgctc cacttcctca aaacttttgg 180ccttacggta aatctcgaga tgaccctaag ggcaagtttt ctgatggcaa aattgtcccg 240gactttattg caaaattcat ggggatacca cacgatttac cgccggcgct aaaacccggc 300accgatgtgt cacgaggagc cagcttcgcc gtcgggtccg cttccattct tggatctcca 360aaagattctt tggctctgaa tcaacaagtg aggaaattca atcagatgat atcaaattgg 420aaagtggatt acattcagaa atcagtgttt atgattagca ttggtatgga agattactac 480aactttacca aaaacaatcc taatgctgaa gtttctgctc aacaagcttt cgttacttct 540gtcactaacc ggtttaagag tgatatcaac ttgttgtatt catctggagc tagtaaattc 600gtcgtacact tgctagcgcc attaggttgt ttaccgatcg caagacaaga atttaaaacc 660ggtaacaatt gttacgagaa actcgatgat ttggccaaac aacacaacgc taaaattgga 720ccgattttga acgaaatggc ggaaactaaa ccggatttcc aattcaccgt tttcgatttc 780tacaacgtta ttcttcgcag gacacaaaga aacatgaact accggttttc cgtgacgaat 840atatcgtgtt gcggtgttgg gacgcattat gcatatggtt gtggtttacc taacgtgcac 900tcgaagttat gcgaatatca aagatcctac ctttacttcg acgcacgtca taacacagag 960aaagcacaag aagcgtttgc tcatcttatc tttggagctg acccaaatgt tatccaacct 1020atgaatgttc gtgagctcat ggtgtatcct gttaatgagc ctatgcgtga gttttgggag 1080gatccaatgg atgagaagtt atcgttagtc caatactag 111912372PRTArabidopsis thaliana 12Met Glu Cys Ser Ser Val Ser Val Leu Gly Ile Leu Leu Val Phe Pro1 5 10 15Leu Leu His Asn Leu Val Thr Ile Ser Gly Gln Asn Leu Pro Ala Val 20 25 30Gly Leu Phe Thr Phe Gly Asp Ser Asn Phe Asp Ala Gly Asn Lys Lys 35 40 45Phe Leu Thr Ser Ala Pro Leu Pro Gln Asn Phe Trp Pro Tyr Gly Lys 50 55 60Ser Arg Asp Asp Pro Lys Gly Lys Phe Ser Asp Gly Lys Ile Val Pro65 70 75 80Asp Phe Ile Ala Lys Phe Met Gly Ile Pro His Asp Leu Pro Pro Ala 85 90 95Leu Lys Pro Gly Thr Asp Val Ser Arg Gly Ala Ser Phe Ala Val Gly 100 105 110Ser Ala Ser Ile Leu Gly Ser Pro Lys Asp Ser Leu Ala Leu Asn Gln 115 120 125Gln Val Arg Lys Phe Asn Gln Met Ile Ser Asn Trp Lys Val Asp Tyr 130 135 140Ile Gln Lys Ser Val Phe Met Ile Ser Ile Gly Met Glu Asp Tyr Tyr145 150 155 160Asn Phe Thr Lys Asn Asn Pro Asn Ala Glu Val Ser Ala Gln Gln Ala 165 170 175Phe Val Thr Ser Val Thr Asn Arg Phe Lys Ser Asp Ile Asn Leu Leu 180 185 190Tyr Ser Ser Gly Ala Ser Lys Phe Val Val His Leu Leu Ala Pro Leu 195 200 205Gly Cys Leu Pro Ile Ala Arg Gln Glu Phe Lys Thr Gly Asn Asn Cys 210 215 220Tyr Glu Lys Leu Asn Asp Leu Ala Lys Gln His Asn Ala Lys Ile Gly225 230 235 240Pro Ile Leu Asn Glu Met Ala Glu Thr Lys Pro Asp Phe Gln Phe Thr 245 250 255Val Phe Asp Phe Tyr Asn Val Ile Leu Arg Arg Thr Gln Arg Asn Met 260 265 270Asn Tyr Arg Phe Ser Val Thr Asn Ile Ser Cys Cys Gly Val Gly Thr 275 280 285His Tyr Ala Tyr Gly Cys Gly Leu Pro Asn Val His Ser Lys Leu Cys 290
295 300Glu Tyr Gln Arg Ser Tyr Leu Tyr Phe Asp Ala Arg His Asn Thr Glu305 310 315 320Lys Ala Gln Glu Ala Phe Ala His Leu Ile Phe Gly Ala Asp Pro Asn 325 330 335Val Ile Gln Pro Met Asn Val Arg Glu Leu Met Val Tyr Pro Val Asn 340 345 350Glu Pro Met Arg Glu Phe Trp Glu Asp Pro Met Asp Glu Lys Leu Ser 355 360 365Leu Val Gln Tyr 37013447DNAHelianthus annuus 13atggttgaaa ccttgtttga agacatattt agggttgatc agcttgatcc agatggcaag 60aagtttgaca aagttaatcg cattgaagca aggagcgatc agttagatat gtacatgcag 120ctggatgtga atacagaggt ttatcctatg catgtcggtg ataagtttat gatggtttta 180gcatctacct taaatttgga tggaactccc gacagtggct tttttactcc gggtggcaga 240aagtctctcg ctgacaaatt tgagtatgtg atgcacggca aattgtacag gatatctgag 300gaagggtccg gagccaatgt taaagcggac atttatgttt cattcggcgg tttattaatg 360ttgttgaggg gcgatccctc aattgcagct aaatttgagc ttgatcagag gttattcatc 420cttatgagga aggtggataa ggcctag 44714148PRTHelianthus annuus 14Met Val Glu Thr Leu Phe Glu Asp Ile Phe Arg Val Asp Gln Leu Asp1 5 10 15Pro Asp Gly Lys Lys Phe Asp Lys Val Asn Arg Ile Glu Ala Arg Ser 20 25 30Asp Gln Leu Asp Met Tyr Met Gln Leu Asp Val Asn Thr Glu Val Tyr 35 40 45Pro Met His Val Gly Asp Lys Phe Met Met Val Leu Ala Ser Thr Leu 50 55 60Asn Leu Asp Gly Thr Pro Asp Ser Gly Phe Phe Thr Pro Gly Gly Arg65 70 75 80Lys Ser Leu Ala Asp Lys Phe Glu Tyr Val Met His Gly Lys Leu Tyr 85 90 95Arg Ile Ser Glu Glu Gly Ser Gly Ala Asn Val Lys Ala Asp Ile Tyr 100 105 110Val Ser Phe Gly Gly Leu Leu Met Leu Leu Arg Gly Asp Pro Ser Ile 115 120 125Ala Ala Lys Phe Glu Leu Asp Gln Arg Leu Phe Ile Leu Met Arg Lys 130 135 140Val Asp Lys Ala14515675DNAHelianthus annuus 15atggttgatg ctgagaaacg gcttttggcg aatgcactga aagaccccga caatcagcat 60tttgtgttac tttctgacag ctgtgtaccg ttgcacaact ttgactatat ttacaactat 120cttatgtaca caaatgtcag cttcattgac agctttgagg atcctggtcc acatggaagt 180ggtcggtact cggatcatat gttacctgaa gtcgaaaaaa aattctttag gaagggtgct 240cagtggttca cgatgaagcg tcaacatgcc atcatagtta tggcggataa tctctactat 300acaaagttca gagattattg taggccgggt atggacgggc gcaattgcta tgcagatgaa 360cattatttgc caacattttt ccatatgttt gatcctactg ggattgcaaa ctggtcggtg 420acacacgttg actggtcaga acgaaaatgg cacccgaaat catacgatct gaaggacgtt 480tcttaccagc tcatcaaaaa tctctcgtct atcactgaaa gtgttcacga aacgagcgat 540agaaagaggg taactacagt tacgccgtgt atgtggaatg gtatgaaccg gccgtgttac 600ttatttgcaa gaaaattcct gcccgagacg ttagacactt tgattgacct tttccctcgt 660tacacgacaa tatga 67516377PRTHelianthus annuus 16Met Gly Lys Lys Asp Met Pro Lys Arg Ser His Leu Lys Lys Pro Thr1 5 10 15Trp Ile Ile Ile Leu Val Ser Leu Val Cys Val Phe Leu Val Val Gly 20 25 30Tyr Val Tyr Pro Pro Arg Asp Ser Thr Ala Cys Tyr Ile Phe Ser Ser 35 40 45Ser Ser Cys Lys Lys Ile Ser Arg Trp Leu Pro Pro Pro Glu Arg Glu 50 55 60Leu Ser Asp Lys Glu Ile Ala Ser Arg Val Val Thr Lys Asn Leu Leu65 70 75 80Asn Thr Pro Pro Ile Lys Thr Lys Asn Pro Lys Ile Ala Phe Met Phe 85 90 95Leu Ser Pro Gly Ser Leu Ala Phe Glu Arg Leu Trp Asp Lys Phe Phe 100 105 110Gln Gly His Glu Gly Arg Phe Ser Ile His Ile His Ala Ser Arg Val 115 120 125Lys Pro Val His Ser Ser Arg Tyr Phe Gln Asn Arg Glu Ile Arg Ser 130 135 140Asp Lys Val Asp Trp Gly Lys Ile Ser Met Val Asp Ala Glu Lys Arg145 150 155 160Leu Leu Ala Asn Ala Leu Lys Asp Pro Asp Asn Gln His Phe Val Leu 165 170 175Leu Ser Asp Ser Cys Val Pro Leu His Asn Phe Asp Tyr Ile Tyr Asn 180 185 190Tyr Leu Met Tyr Thr Asn Val Ser Phe Ile Asp Ser Phe Glu Asp Pro 195 200 205Gly Pro His Gly Ser Gly Arg Tyr Ser Asp His Met Leu Pro Glu Val 210 215 220Glu Lys Lys Phe Phe Arg Lys Gly Ala Gln Trp Phe Thr Met Lys Arg225 230 235 240Gln His Ala Ile Ile Val Met Ala Asp Asn Leu Tyr Tyr Thr Lys Phe 245 250 255Arg Asp Tyr Cys Arg Pro Gly Met Asp Gly Arg Asn Cys Tyr Ala Asp 260 265 270Glu His Tyr Leu Pro Thr Phe Phe His Met Phe Asp Pro Thr Gly Ile 275 280 285Ala Asn Trp Ser Val Thr His Val Asp Trp Ser Glu Arg Lys Trp His 290 295 300Pro Lys Ser Tyr Asp Leu Lys Asp Val Ser Tyr Gln Leu Ile Lys Asn305 310 315 320Leu Ser Ser Ile Thr Glu Ser Val His Glu Thr Ser Asp Arg Lys Arg 325 330 335Val Thr Thr Val Thr Pro Cys Met Trp Asn Gly Met Asn Arg Pro Cys 340 345 350Tyr Leu Phe Ala Arg Lys Phe Leu Pro Glu Thr Leu Asp Thr Leu Ile 355 360 365Asp Leu Phe Pro Arg Tyr Thr Thr Ile 370 37517594DNAHelianthus annuus 17atgatattat cgtttcgtgg acaagttgaa ttattggcaa aacatcctta cggttgccgt 60gtcatacaga gggtattaga gcattcaacg gacgaagtac aaagccgatt catagtggac 120gagatcttgg agaatgttta tgttcttgca caagatcagt atggcaatta tgtaactcag 180tatgtgttgg aggcggagaa accagaggtg agaagccaga tagtagacaa attgttgggc 240catatagtgc gattaagcca acacaaatat gcctcaaatg ttgtcgaaaa atgtttggaa 300tatggtgatg aagctataag aaaaatccta attgaagaga ttattgaatg tgctgatggc 360aatgataact tattggtgtt ggtgaaagac caatatgcaa attatgtggt ccaaaaggtt 420cttcaaatat gtagcgacca ccaacggaaa gtgttgctta gtcgtatgaa aggtcatctg 480aatttgttga agacatatac ttatgggaaa catatcgttg ctcgctttga acaattgtat 540ggtgaagaaa ttgcggcgtt gggttcaaac atgagcgatg gcaaatctgt atag 59418197PRTHelianthus annuus 18Met Ile Leu Ser Phe Arg Gly Gln Val Glu Leu Leu Ala Lys His Pro1 5 10 15Tyr Gly Cys Arg Val Ile Gln Arg Val Leu Glu His Ser Thr Asp Glu 20 25 30Val Gln Ser Arg Phe Ile Val Asp Glu Ile Leu Glu Asn Val Tyr Val 35 40 45Leu Ala Gln Asp Gln Tyr Gly Asn Tyr Val Thr Gln Tyr Val Leu Glu 50 55 60Ala Glu Lys Pro Glu Val Arg Ser Gln Ile Val Asp Lys Leu Leu Gly65 70 75 80His Ile Val Arg Leu Ser Gln His Lys Tyr Ala Ser Asn Val Val Glu 85 90 95Lys Cys Leu Glu Tyr Gly Asp Glu Ala Ile Arg Lys Ile Leu Ile Glu 100 105 110Glu Ile Ile Glu Cys Ala Asp Gly Asn Asp Asn Leu Leu Val Leu Val 115 120 125Lys Asp Gln Tyr Ala Asn Tyr Val Val Gln Lys Val Leu Gln Ile Cys 130 135 140Ser Asp His Gln Arg Lys Val Leu Leu Ser Arg Met Lys Gly His Leu145 150 155 160Asn Leu Leu Lys Thr Tyr Thr Tyr Gly Lys His Ile Val Ala Arg Phe 165 170 175Glu Gln Leu Tyr Gly Glu Glu Ile Ala Ala Leu Gly Ser Asn Met Ser 180 185 190Asp Gly Lys Ser Val 19519780DNAHelianthus annuus 19atgaaccgga tgatcgaagc gtggaatgcg attccggatg tgccggagtc acattcttct 60aaaggtgata tggcagaaga atctgatcat atacaaaatc caccaaaaac accttatata 120atgagtaagc gaactacaac gagaagaaat tcgcttgaga atagcagcag gaaaactgtt 180ccagccatgt tcagtaagct ggatcgcaag aaacccgtta atcagaaact cgacactgct 240gaggttcata tgaaacacga aacaaaacgg gccgttttta gtgaaatttc tgatgaaaag 300atgcaagaat ctcgatataa tgaagataga tcgagttcca aagttgtggg gagcaatggc 360ggattgaata ccaactctgt tgaacaagaa tccgaggact tatctttgat ccgcaatcaa 420cttgctcaaa tcgagaccca acaatccaat ttatatgatc tcctcgagaa atttatcggg 480agctcgctga acgggatgca atctctggag tctcgtgtgc gcggtgtaga gtcaacactc 540gacgagattt catttgactt agcaaagtca actggacagg tgtcgaatcc ggaacccacc 600ttgtgttgta agttaccagg tgcagatctt attagctcta aactctggaa gaaatcagaa 660atccaacagt acccatctgt caagaacaag gatcttgaat cattcaactt tcaaaccacc 720ggattccatc tccgtgccgg gctaattaag aacccattgg ccgaggtgca ccaaatatga 78020259PRTHelianthus annuus 20Met Asn Arg Met Ile Glu Ala Trp Asn Ala Ile Pro Asp Val Pro Glu1 5 10 15Ser His Ser Ser Lys Gly Asp Met Ala Glu Glu Ser Asp His Ile Gln 20 25 30Asn Pro Pro Lys Thr Pro Tyr Ile Met Ser Lys Arg Thr Thr Thr Arg 35 40 45Arg Asn Ser Leu Glu Asn Ser Ser Arg Lys Thr Val Pro Ala Met Phe 50 55 60Ser Lys Leu Asp Arg Lys Lys Pro Val Asn Gln Lys Leu Asp Thr Ala65 70 75 80Glu Val His Met Lys His Glu Thr Lys Arg Ala Val Phe Ser Glu Ile 85 90 95Ser Asp Glu Lys Met Gln Glu Ser Arg Tyr Asn Glu Asp Arg Ser Ser 100 105 110Ser Lys Val Val Gly Ser Asn Gly Gly Leu Asn Thr Asn Ser Val Glu 115 120 125Gln Glu Ser Glu Asp Leu Ser Leu Ile Arg Asn Gln Leu Ala Gln Ile 130 135 140Glu Thr Gln Gln Ser Asn Leu Tyr Asp Leu Leu Glu Lys Phe Ile Gly145 150 155 160Ser Ser Leu Asn Gly Met Gln Ser Leu Glu Ser Arg Val Arg Gly Val 165 170 175Glu Ser Thr Leu Asp Glu Ile Ser Phe Asp Leu Ala Lys Ser Thr Gly 180 185 190Gln Val Ser Asn Pro Glu Pro Thr Leu Cys Cys Lys Leu Pro Gly Ala 195 200 205Asp Leu Ile Ser Ser Lys Leu Trp Lys Lys Ser Glu Ile Gln Gln Tyr 210 215 220Pro Ser Val Lys Asn Lys Asp Leu Glu Ser Phe Asn Phe Gln Thr Thr225 230 235 240Gly Phe His Leu Arg Ala Gly Leu Ile Lys Asn Pro Leu Ala Glu Val 245 250 255His Gln Ile21915DNAHelianthus annuus 21atgcagaccg tttctcggag attagctcgt gaaaatttga gctctcgcac atcgatttac 60tctctcaaat cgctttatcc tgtttccgat cgctgttacg gtgagtatga tcggcgttat 120gcctctacgc ttaccaccaa aggtattgga catctggtcc gcaagggtac tggtggaaga 180tcgtctgtta gtgggatagt tgctacagta ttcggagcta ctggtttcct tgggcgttac 240ttggtgcaac agcttgctaa aacgggttca caagtgctag taccatttag aggttccgaa 300gattcgcccc gtcatctcaa actgatgggc gatttggggc agattgttcc catgaaatat 360aatcctagag atgaaaactc aattaaggca gtcatggcca aggcaaatgt tgtgattaat 420ctcataggaa gggaatatga aaccagaaat tatagttttg aggaagtgaa ccatcatatg 480gctgaacaac ttgcaaagat ttccaaagaa catggtggaa tcatgagatt tatacaactg 540tcgtgtttag gtgcatctaa atcatctcca tctaggatgc ttcaagccaa ggctgctgca 600gaagaatcca tcttacgtga attgcctgag gccacaatac tgcgacctgc agtgatggtt 660ggtacagaag atcggatctt gaacccatgg gckcagatcg ctaaaaaata taactttctt 720ccaatgatcg gggswgrytc tactaagatt cagccatggt atgttgctga tgtcgcctct 780gcagttgttg cggcactaag tgatgacggg agtagcacgg aaaaagagta tgacactatg 840gtgggcctga tagtttatac actgcatcaa ctggctgaac ttatgtatca aacgattcga 900gaatggactc attga 91522304PRTHelianthus annuusmisc_feature(233)..(233)Xaa can be any naturally occurring amino acid 22Met Gln Thr Val Ser Arg Arg Leu Ala Arg Glu Asn Leu Ser Ser Arg1 5 10 15Thr Ser Ile Tyr Ser Leu Lys Ser Leu Tyr Pro Val Ser Asp Arg Cys 20 25 30Tyr Gly Glu Tyr Asp Arg Arg Tyr Ala Ser Thr Leu Thr Thr Lys Gly 35 40 45Ile Gly His Leu Val Arg Lys Gly Thr Gly Gly Arg Ser Ser Val Ser 50 55 60Gly Ile Val Ala Thr Val Phe Gly Ala Thr Gly Phe Leu Gly Arg Tyr65 70 75 80Leu Val Gln Gln Leu Ala Lys Thr Gly Ser Gln Val Leu Val Pro Phe 85 90 95Arg Gly Ser Glu Asp Ser Pro Arg His Leu Lys Leu Met Gly Asp Leu 100 105 110Gly Gln Ile Val Pro Met Lys Tyr Asn Pro Arg Asp Glu Asn Ser Ile 115 120 125Lys Ala Val Met Ala Lys Ala Asn Val Val Ile Asn Leu Ile Gly Arg 130 135 140Glu Tyr Glu Thr Arg Asn Tyr Ser Phe Glu Glu Val Asn His His Met145 150 155 160Ala Glu Gln Leu Ala Lys Ile Ser Lys Glu His Gly Gly Ile Met Arg 165 170 175Phe Ile Gln Leu Ser Cys Leu Gly Ala Ser Lys Ser Ser Pro Ser Arg 180 185 190Met Leu Gln Ala Lys Ala Ala Ala Glu Glu Ser Ile Leu Arg Glu Leu 195 200 205Pro Glu Ala Thr Ile Leu Arg Pro Ala Val Met Val Gly Thr Glu Asp 210 215 220Arg Ile Leu Asn Pro Trp Ala Gln Xaa Ala Lys Lys Tyr Asn Phe Leu225 230 235 240Pro Met Ile Gly Xaa Xaa Ser Thr Lys Ile Gln Pro Trp Tyr Val Ala 245 250 255Asp Val Ala Ser Ala Val Val Ala Ala Leu Ser Asp Asp Gly Ser Ser 260 265 270Thr Glu Lys Glu Tyr Asp Thr Met Val Gly Leu Ile Val Tyr Thr Leu 275 280 285His Gln Leu Ala Glu Leu Met Tyr Gln Thr Ile Arg Glu Trp Thr His 290 295 300231521DNAArabidopsis thaliana 23atgaagatat actctagaac ggttgctgtt tcactcattg tgtcattcct cctgtgtttc 60tctgcctttg ctgagcgcaa tgacgggacc ttcagagttg gactgaaaaa actcaagttg 120gattcgaaaa atcggcttgc agcacgcgtc gaatccaagc aagaaaagcc cctgagagct 180tacagacttg gagattctgg agatgctgat gttgttgtgc ttaagaatta tctagatgct 240cagtactatg gtgagatcgc cattggtact ccacctcaga agttcactgt ggtttttgac 300actgggagct ctaacctctg ggtgccatca tcaaaatgct atttctcact tgcatgtctc 360ttgcatccca aatacaagtc gtctcgttca agcacatatg agaagaatgg aaaagctgcc 420gcaattcatt acggcactgg agcaattgct ggttttttta gtaatgatgc tgtcacagtt 480ggcgatttag ttgtcaagga tcaggagttt atcgaggcaa ccaaggagcc tggtataaca 540tttgttgtag ctaaatttga tggtatcctt ggtcttggat tccaagagat ctctgttgga 600aaagctgctc ctgtttggta caacatgctc aagcaaggcc ttatcaagga gccggttttt 660tcattttggc ttaaccgtaa tgcagatgaa gaagaaggtg gtgaacttgt atttggaggt 720gttgatccaa atcatttcaa gggcaaacat acatatgttc ctgtgacaca aaagggctac 780tggcagtttg acatgggtga tgttcttatt ggcggtgcac ccactgggtt ctgtgaaagt 840ggctgttctg cgatagcaga ttctggtaca tctttgcttg ccggtccaac gactataatc 900accatgataa accatgctat tggagcagct ggagttgtta gccagcagtg caagactgtt 960gtggatcaat acgggcagac cattttggat ttacttttgt ctgagaccca accgaagaaa 1020atctgctcgc agattggtct gtgtactttt gatggtaccc gtggtgtcag tatgggcatt 1080gagtcggtgg tggacaagga aaacgccaaa ttgtctaatg gtgttggaga tgctgcgtgt 1140tctgcatgtg agatggctgt tgtgtggatc cagagccagt tgaggcaaaa catgactcaa 1200gagcgcatat tgaactacgt caacgagcta tgtgaacgcc tccccagccc aatgggagag 1260tctgcagttg actgtgcaca actgtcaaca atgcccactg tttcacttac cattggaggc 1320aaagtatttg atcttgctcc agaagagtat gttctgaaag ttggtgaggg acctgtggca 1380cagtgcatca gtggttttat tgctcttgac gttgctccac ctcgtggacc tctctggatc 1440ttgggagatg tgttcatggg caaataccac accgtatttg actttggtaa cgaacaggtc 1500gggtttgcag aggcagccta a 152124506PRTArabidopsis thaliana 24Met Lys Ile Tyr Ser Arg Thr Val Ala Val Ser Leu Ile Val Ser Phe1 5 10 15Leu Leu Cys Phe Ser Ala Phe Ala Glu Arg Asn Asp Gly Thr Phe Arg 20 25 30Val Gly Leu Lys Lys Leu Lys Leu Asp Ser Lys Asn Arg Leu Ala Ala 35 40 45Arg Val Glu Ser Lys Gln Glu Lys Pro Leu Arg Ala Tyr Arg Leu Gly 50 55 60Asp Ser Gly Asp Ala Asp Val Val Val Leu Lys Asn Tyr Leu Asp Ala65 70 75 80Gln Tyr Tyr Gly Glu Ile Ala Ile Gly Thr Pro Pro Gln Lys Phe Thr 85 90 95Val Val Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ser Lys 100 105 110Cys Tyr Phe Ser Leu Ala Cys Leu Leu His Pro Lys Tyr Lys Ser Ser 115 120 125Arg Ser Ser Thr Tyr Glu Lys Asn Gly Lys Ala Ala Ala Ile His Tyr 130 135 140Gly Thr Gly Ala Ile Ala Gly Phe Phe Ser Asn Asp Ala Val Thr Val145 150 155 160Gly Asp Leu Val Val Lys Asp Gln Glu Phe Ile Glu Ala Thr Lys Glu 165 170 175Pro Gly Ile Thr Phe Val Val Ala Lys Phe Asp Gly Ile Leu Gly Leu 180 185 190Gly Phe Gln Glu Ile Ser Val Gly Lys Ala Ala Pro Val Trp Tyr Asn 195 200 205Met Leu Lys Gln Gly Leu Ile Lys Glu Pro Val Phe Ser Phe Trp Leu 210 215
220Asn Arg Asn Ala Asp Glu Glu Glu Gly Gly Glu Leu Val Phe Gly Gly225 230 235 240Val Asp Pro Asn His Phe Lys Gly Lys His Thr Tyr Val Pro Val Thr 245 250 255Gln Lys Gly Tyr Trp Gln Phe Asp Met Gly Asp Val Leu Ile Gly Gly 260 265 270Ala Pro Thr Gly Phe Cys Glu Ser Gly Cys Ser Ala Ile Ala Asp Ser 275 280 285Gly Thr Ser Leu Leu Ala Gly Pro Thr Thr Ile Ile Thr Met Ile Asn 290 295 300His Ala Ile Gly Ala Ala Gly Val Val Ser Gln Gln Cys Lys Thr Val305 310 315 320Val Asp Gln Tyr Gly Gln Thr Ile Leu Asp Leu Leu Leu Ser Glu Thr 325 330 335Gln Pro Lys Lys Ile Cys Ser Gln Ile Gly Leu Cys Thr Phe Asp Gly 340 345 350Thr Arg Gly Val Ser Met Gly Ile Glu Ser Val Val Asp Lys Glu Asn 355 360 365Ala Lys Leu Ser Asn Gly Val Gly Asp Ala Ala Cys Ser Ala Cys Glu 370 375 380Met Ala Val Val Trp Ile Gln Ser Gln Leu Arg Gln Asn Met Thr Gln385 390 395 400Glu Arg Ile Leu Asn Tyr Val Asn Glu Leu Cys Glu Arg Leu Pro Ser 405 410 415Pro Met Gly Glu Ser Ala Val Asp Cys Ala Gln Leu Ser Thr Met Pro 420 425 430Thr Val Ser Leu Thr Ile Gly Gly Lys Val Phe Asp Leu Ala Pro Glu 435 440 445Glu Tyr Val Leu Lys Val Gly Glu Gly Pro Val Ala Gln Cys Ile Ser 450 455 460Gly Phe Ile Ala Leu Asp Val Ala Pro Pro Arg Gly Pro Leu Trp Ile465 470 475 480Leu Gly Asp Val Phe Met Gly Lys Tyr His Thr Val Phe Asp Phe Gly 485 490 495Asn Glu Gln Val Gly Phe Ala Glu Ala Ala 500 505251542DNAArabidopsis thaliana 25atgggagtat actcgagagc ggttgcgttt tcgggctttg tgtcgtttct gctgtttttc 60actgcttatt ctaagagaaa tgatggaaca ttcagagttg gcctgaaaaa actgaagttg 120gatcctaaca accgactcgc aacacgcttt ggttccaagc aagaagaggc cttgagatct 180tctttgcgtt cgtacaacaa caatcttggt ggtgattctg gagatgctga tattgtcccg 240ctcaagaatt acttggatgc tcagtactat ggtgagattg ctattggtac tccaccgcag 300aagttcactg tcatttttga taccggaagc tctaaccttt gggtgccatc aggaaaatgt 360tttttctcgc tgtcttgtta ctttcatgct aagtacaagt cctcgcgatc aagcacatat 420aagaagagtg gaaaacgtgc cgcaatccat tacggctcag gatcaatctc tggtttcttt 480agttatgatg ctgtcacggt tggtgatttg gttgtcaaag atcaggagtt tattgagaca 540accagtgagc ctggtttaac attcctggtg gctaagtttg atggtcttct tggtcttggg 600ttccaagaga tcgctgttgg aaacgctact cctgtttggt acaatatgct caagcaaggc 660cttataaaga ggccggtctt ttcattttgg cttaaccgtg atccaaagag tgaagaaggc 720ggtgaaatcg tattcggagg tgttgatcca aagcatttta gaggagaaca tacatttgtt 780cctgtgacac aaaggggtta ctggcagttc gacatgggtg aggttctcat tgccggtgaa 840tctactggat attgtggaag tggttgttct gcgatagcag attctggaac atcgttactt 900gcgggtccaa cggctgtggt tgccatgata aataaggcta ttggagcatc tggagttgtt 960agccagcagt gcaagactgt tgttgaccag tatggacaaa ccattttgga tttacttttg 1020gctgagactc aaccaaagaa gatttgctca caaattggtc tttgcgctta cgatggcacc 1080catggggtca gtatggggat tgaatcggtg gtggacaagg aaaacacaag atcatctagt 1140ggtcttcgag acgcgggttg tcctgcatgt gaaatggcgg ttgtgtggat acagagccaa 1200ttgaggcaga acatgactca agagaggata gtgaactaca ttaatgagat atgcgagcgc 1260atgcctagtc caaatggaga gtctgctgtt gactgctcac aactttctaa aatgcctact 1320gtttcattca ccattggaga caaagtcttt gatcttgctc ccgaagagta cgtactgaag 1380attggggaag gaccagtggc acaatgtatt agcggcttta ccgcacttga tatccctcca 1440cctcgtggac ctctctggat acttggagat gtgtttatgg gaaaatacca cactgtcttt 1500gacttcggaa acgagcaggt tggcttcgca gaagccgtgt ga 154226513PRTArabidopsis thaliana 26Met Gly Val Tyr Ser Arg Ala Val Ala Phe Ser Gly Phe Val Ser Phe1 5 10 15Leu Leu Phe Phe Thr Ala Tyr Ser Lys Arg Asn Asp Gly Thr Phe Arg 20 25 30Val Gly Leu Lys Lys Leu Lys Leu Asp Pro Asn Asn Arg Leu Ala Thr 35 40 45Arg Phe Gly Ser Lys Gln Glu Glu Ala Leu Arg Ser Ser Leu Arg Ser 50 55 60Tyr Asn Asn Asn Leu Gly Gly Asp Ser Gly Asp Ala Asp Ile Val Pro65 70 75 80Leu Lys Asn Tyr Leu Asp Ala Gln Tyr Tyr Gly Glu Ile Ala Ile Gly 85 90 95Thr Pro Pro Gln Lys Phe Thr Val Ile Phe Asp Thr Gly Ser Ser Asn 100 105 110Leu Trp Val Pro Ser Gly Lys Cys Phe Phe Ser Leu Ser Cys Tyr Phe 115 120 125His Ala Lys Tyr Lys Ser Ser Arg Ser Ser Thr Tyr Lys Lys Ser Gly 130 135 140Lys Arg Ala Ala Ile His Tyr Gly Ser Gly Ser Ile Ser Gly Phe Phe145 150 155 160Ser Tyr Asp Ala Val Thr Val Gly Asp Leu Val Val Lys Asp Gln Glu 165 170 175Phe Ile Glu Thr Thr Ser Glu Pro Gly Leu Thr Phe Leu Val Ala Lys 180 185 190Phe Asp Gly Leu Leu Gly Leu Gly Phe Gln Glu Ile Ala Val Gly Asn 195 200 205Ala Thr Pro Val Trp Tyr Asn Met Leu Lys Gln Gly Leu Ile Lys Arg 210 215 220Pro Val Phe Ser Phe Trp Leu Asn Arg Asp Pro Lys Ser Glu Glu Gly225 230 235 240Gly Glu Ile Val Phe Gly Gly Val Asp Pro Lys His Phe Arg Gly Glu 245 250 255His Thr Phe Val Pro Val Thr Gln Arg Gly Tyr Trp Gln Phe Asp Met 260 265 270Gly Glu Val Leu Ile Ala Gly Glu Ser Thr Gly Tyr Cys Gly Ser Gly 275 280 285Cys Ser Ala Ile Ala Asp Ser Gly Thr Ser Leu Leu Ala Gly Pro Thr 290 295 300Ala Val Val Ala Met Ile Asn Lys Ala Ile Gly Ala Ser Gly Val Val305 310 315 320Ser Gln Gln Cys Lys Thr Val Val Asp Gln Tyr Gly Gln Thr Ile Leu 325 330 335Asp Leu Leu Leu Ala Glu Thr Gln Pro Lys Lys Ile Cys Ser Gln Ile 340 345 350Gly Leu Cys Ala Tyr Asp Gly Thr His Gly Val Ser Met Gly Ile Glu 355 360 365Ser Val Val Asp Lys Glu Asn Thr Arg Ser Ser Ser Gly Leu Arg Asp 370 375 380Ala Gly Cys Pro Ala Cys Glu Met Ala Val Val Trp Ile Gln Ser Gln385 390 395 400Leu Arg Gln Asn Met Thr Gln Glu Arg Ile Val Asn Tyr Ile Asn Glu 405 410 415Ile Cys Glu Arg Met Pro Ser Pro Asn Gly Glu Ser Ala Val Asp Cys 420 425 430Ser Gln Leu Ser Lys Met Pro Thr Val Ser Phe Thr Ile Gly Asp Lys 435 440 445Val Phe Asp Leu Ala Pro Glu Glu Tyr Val Leu Lys Ile Gly Glu Gly 450 455 460Pro Val Ala Gln Cys Ile Ser Gly Phe Thr Ala Leu Asp Ile Pro Pro465 470 475 480Pro Arg Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Met Gly Lys Tyr 485 490 495His Thr Val Phe Asp Phe Gly Asn Glu Gln Val Gly Phe Ala Glu Ala 500 505 510Val 271527DNAArabidopsis thaliana 27atgggaacta ggttccaatc attcttgctc gtgttcttgc tttcatgttt aatcctcata 60tcaactgcct cgtgtgagcg aaatggtgat ggaacgatta gaattggatt gaagaagagg 120aaactagacc ggagcaacag gctagcttct cagctttttt tgaaaaaccg agggtctcat 180tggtctccca aacactattt tcgcctgaac gatgaaaatg cggatatggt tccgctgaaa 240aactatttgg atgctcaata ctatggtgac attaccattg gtactcctcc tcagaagttc 300actgtgatct ttgatactgg tagctccaat ctctggatac catctactaa atgttaccta 360tcggttgctt gttatttcca ctccaagtac aaggctagcc agtcatcgtc atatagaaag 420aacggaaaac ccgcgtctat ccgctatggg acgggtgcta tttctggtta ctttagcaat 480gatgatgtta aagttggtga tattgttgtg aaagagcagg aattcataga ggctactagt 540gagcctggta taacattctt gctagcaaag tttgatggta tcctcggttt gggtttcaaa 600gagatttctg taggaaactc aactccggtt tggtataaca tggtagaaaa aggtttagtt 660aaggaaccga ttttttcgtt ctggcttaac cgtaacccga aagatccaga aggcggtgag 720attgttttcg gtggagtcga cccgaagcac ttcaaaggag agcatacttt tgttcctgtg 780acacataaag gttactggca gtttgacatg ggtgatctcc aaattgctgg caaaccaacc 840ggatattgtg ctaaaggttg ttctgctatt gctgattccg gaacttctct gctcaccggt 900ccatcgactg tcatcacgat gatcaatcac gcgataggag cacaaggaat tgtaagccgt 960gaatgcaagg ccgtggtgga tcaatacgga aaaaccatgt tgaattctct tctagctcag 1020gaggatccga agaaagtatg ctcacaaatt ggagtctgcg cttatgatgg tacacagagt 1080gtgagtatgg ggatccggtc agttgtagac gatggaacat cgggtctttt aaaccaagcg 1140atgtgcagtg cttgcgaaat ggcagccgtg tggatggaga gcgaattgac tcagaatcaa 1200acacaagaac gcatactcgc ttatgctgct gagctatgtg accatatacc aacccaaaac 1260caacaatcag cagtggactg tgggagggtt tcgtcgatgc ctatagtcac attctcaatt 1320ggtggcagaa gctttgatct aactcctcaa gaccatatat tcaagatcgg ggaaggagtt 1380gagtctcagt gcaccagcgg tttcacggca atggatattg ctccgcctcg tggacctctc 1440tggatcttgg gtgatatctt catgggacca taccacacag tgttcgatta tgggaaagga 1500agagttggat tcgccaaagc tgcttaa 152728508PRTArabidopsis thaliana 28Met Gly Thr Arg Phe Gln Ser Phe Leu Leu Val Phe Leu Leu Ser Cys1 5 10 15Leu Ile Leu Ile Ser Thr Ala Ser Cys Glu Arg Asn Gly Asp Gly Thr 20 25 30Ile Arg Ile Gly Leu Lys Lys Arg Lys Leu Asp Arg Ser Asn Arg Leu 35 40 45Ala Ser Gln Leu Phe Leu Lys Asn Arg Gly Ser His Trp Ser Pro Lys 50 55 60His Tyr Phe Arg Leu Asn Asp Glu Asn Ala Asp Met Val Pro Leu Lys65 70 75 80Asn Tyr Leu Asp Ala Gln Tyr Tyr Gly Asp Ile Thr Ile Gly Thr Pro 85 90 95Pro Gln Lys Phe Thr Val Ile Phe Asp Thr Gly Ser Ser Asn Leu Trp 100 105 110Ile Pro Ser Thr Lys Cys Tyr Leu Ser Val Ala Cys Tyr Phe His Ser 115 120 125Lys Tyr Lys Ala Ser Gln Ser Ser Ser Tyr Arg Lys Asn Gly Lys Pro 130 135 140Ala Ser Ile Arg Tyr Gly Thr Gly Ala Ile Ser Gly Tyr Phe Ser Asn145 150 155 160Asp Asp Val Lys Val Gly Asp Ile Val Val Lys Glu Gln Glu Phe Ile 165 170 175Glu Ala Thr Ser Glu Pro Gly Ile Thr Phe Leu Leu Ala Lys Phe Asp 180 185 190Gly Ile Leu Gly Leu Gly Phe Lys Glu Ile Ser Val Gly Asn Ser Thr 195 200 205Pro Val Trp Tyr Asn Met Val Glu Lys Gly Leu Val Lys Glu Pro Ile 210 215 220Phe Ser Phe Trp Leu Asn Arg Asn Pro Lys Asp Pro Glu Gly Gly Glu225 230 235 240Ile Val Phe Gly Gly Val Asp Pro Lys His Phe Lys Gly Glu His Thr 245 250 255Phe Val Pro Val Thr His Lys Gly Tyr Trp Gln Phe Asp Met Gly Asp 260 265 270Leu Gln Ile Ala Gly Lys Pro Thr Gly Tyr Cys Ala Lys Gly Cys Ser 275 280 285Ala Ile Ala Asp Ser Gly Thr Ser Leu Leu Thr Gly Pro Ser Thr Val 290 295 300Ile Thr Met Ile Asn His Ala Ile Gly Ala Gln Gly Ile Val Ser Arg305 310 315 320Glu Cys Lys Ala Val Val Asp Gln Tyr Gly Lys Thr Met Leu Asn Ser 325 330 335Leu Leu Ala Gln Glu Asp Pro Lys Lys Val Cys Ser Gln Ile Gly Val 340 345 350Cys Ala Tyr Asp Gly Thr Gln Ser Val Ser Met Gly Ile Arg Ser Val 355 360 365Val Asp Asp Gly Thr Ser Gly Leu Leu Asn Gln Ala Met Cys Ser Ala 370 375 380Cys Glu Met Ala Ala Val Trp Met Glu Ser Glu Leu Thr Gln Asn Gln385 390 395 400Thr Gln Glu Arg Ile Leu Ala Tyr Ala Ala Glu Leu Cys Asp His Ile 405 410 415Pro Thr Gln Asn Gln Gln Ser Ala Val Asp Cys Gly Arg Val Ser Ser 420 425 430Met Pro Ile Val Thr Phe Ser Ile Gly Gly Arg Ser Phe Asp Leu Thr 435 440 445Pro Gln Asp His Ile Phe Lys Ile Gly Glu Gly Val Glu Ser Gln Cys 450 455 460Thr Ser Gly Phe Thr Ala Met Asp Ile Ala Pro Pro Arg Gly Pro Leu465 470 475 480Trp Ile Leu Gly Asp Ile Phe Met Gly Pro Tyr His Thr Val Phe Asp 485 490 495Tyr Gly Lys Gly Arg Val Gly Phe Ala Lys Ala Ala 500 50529951DNAArabidopsis thaliana 29atggagaaag gtttgacgat gtcttgtgtt ttggtggtgg ttgcattctt agccatggtt 60catgtctctg tttcagttcc gttcgtagtg tttcctgaaa tcggaacaca atgttctgat 120gctccaaatg ctaacttcac acagcttctc agtaacctct ctagctcacc tggcttttgc 180atagaaattg gcgagggaaa tccaataggc gcttcatggt taataccact tacacaacaa 240gcggaagtag cgtgtgataa ggtgacgcag atggaagagt tgagtcaagg atacaacatt 300gttggaagag ctcaggggag cttagtggct cgaggcttaa tcgagttctg cgaaggtggg 360cctcctgttc acaactatat atccttggct ggtcctcatg ctggcaccgc cgatcttctt 420cggtgtaata cttctggctt aatttgtgac atagcaaatg ggataggcaa ggaaaatccc 480tacagcgact ttgttcaaga taatcttgct cctagtggtt atttcaaaaa ccctaaaaat 540gtgacagggt acctgaaaga ctgtcagtat ctacctaagc ttaacaatga gagaccatac 600gaaagaaaca caacttacaa agaccgtttc gcaagtttac agaacctggt ttttgtcctg 660tttgagaacg atacggttat tgttccaaaa gagtcatctt ggttcgggtt ttatccggat 720ggtgacttaa cacatgttct ccctgttcaa gagacaaagc tctatataga agattggata 780ggtctgaaag cattggttgt tgctggaaaa gtgcagtttg tgaatgtaac cggtgaccac 840ttaataatgg cggacgaaga tctcgtcaaa tacgtcgtac ctcttctcca ggatcaacag 900tctgccccac caagactcaa ccgcaagacc aaggagccct tgcatcctta a 95130316PRTArabidopsis thaliana 30Met Glu Lys Gly Leu Thr Met Ser Cys Val Leu Val Val Val Ala Phe1 5 10 15Leu Ala Met Val His Val Ser Val Ser Val Pro Phe Val Val Phe Pro 20 25 30Glu Ile Gly Thr Gln Cys Ser Asp Ala Pro Asn Ala Asn Phe Thr Gln 35 40 45Leu Leu Ser Asn Leu Ser Ser Ser Pro Gly Phe Cys Ile Glu Ile Gly 50 55 60Glu Gly Asn Pro Ile Gly Ala Ser Trp Leu Ile Pro Leu Thr Gln Gln65 70 75 80Ala Glu Val Ala Cys Asp Lys Val Thr Gln Met Glu Glu Leu Ser Gln 85 90 95Gly Tyr Asn Ile Val Gly Arg Ala Gln Gly Ser Leu Val Ala Arg Gly 100 105 110Leu Ile Glu Phe Cys Glu Gly Gly Pro Pro Val His Asn Tyr Ile Ser 115 120 125Leu Ala Gly Pro His Ala Gly Thr Ala Asp Leu Leu Arg Cys Asn Thr 130 135 140Ser Gly Leu Ile Cys Asp Ile Ala Asn Gly Ile Gly Lys Glu Asn Pro145 150 155 160Tyr Ser Asp Phe Val Gln Asp Asn Leu Ala Pro Ser Gly Tyr Phe Lys 165 170 175Asn Pro Lys Asn Val Thr Gly Tyr Leu Lys Asp Cys Gln Tyr Leu Pro 180 185 190Lys Leu Asn Asn Glu Arg Pro Tyr Glu Arg Asn Thr Thr Tyr Lys Asp 195 200 205Arg Phe Ala Ser Leu Gln Asn Leu Val Phe Val Leu Phe Glu Asn Asp 210 215 220Thr Val Ile Val Pro Lys Glu Ser Ser Trp Phe Gly Phe Tyr Pro Asp225 230 235 240Gly Asp Leu Thr His Val Leu Pro Val Gln Glu Thr Lys Leu Tyr Ile 245 250 255Glu Asp Trp Ile Gly Leu Lys Ala Leu Val Val Ala Gly Lys Val Gln 260 265 270Phe Val Asn Val Thr Gly Asp His Leu Ile Met Ala Asp Glu Asp Leu 275 280 285Val Lys Tyr Val Val Pro Leu Leu Gln Asp Gln Gln Ser Ala Pro Pro 290 295 300Arg Leu Asn Arg Lys Thr Lys Glu Pro Leu His Pro305 310 315311473DNAArabidopsis thaliana 31atgcattggc atggtgtaga gcagccgaga aatccgtggt cagatggtcc tgaatacatc 60acacaatgcc cgattcgacc cgggtcagat tttttgtaca aagtcatatt ttccatcgaa 120gacacgactg tttggtggca cgcgcatagc tcttggacac gtgcaactgt acacggtctt 180attttcgtat atcctcgccc cccgcaaatc ctcccttttc caaaggcaga ccatgaagtc 240cccataattt tgggtgagtg gtggaagagg gatgtgagag aggtcgttga ggagttcgta 300aggacaggag gggctcctaa tgtgtccgat gctttgacca tcaatggaca tcctggtttc 360ttgtatcctt gctctaaatc agatacattt catctcacgg tagagaaggg gaaaacctac 420cgcattcgga tggtaaatgc cgcaatgaac ctacctctct ttttcgcaat cgcgaaccac 480agcctcaccg tagtctcggc cgatggacac tacatcaaac ctataaaggc tacttatatc 540actatatccc ctggcgaaac actagacatg ttattacacg ctgaccaaga ccccgaacgc 600acttattaca tggctgccag agcttaccaa agcggaaata tcgatttcaa caactccact 660accataggaa tcttaagtta cacctcttca tgcaaagcta aaacatcatc gttttcagga 720tattacccaa cccttccttt ttacaatgac acctcagcag cttttggatt ctttaccaag 780atcaaatgct tattctccgg tcaagttcct gtccaaatct ctcgtaggat aatcacgacg 840gtttcaataa atcttcgcat gtgtcctcaa aactcgtgcg aaggtccaaa tgggtcgaga 900ttagcagcga
gtatgaacaa catatcgttt gtcacaccaa gtcacgtgga catactaaaa 960gcttattact atcacattaa aggcgtttat ggaacgcggt ttccggagtt tccaccgttg 1020atttttaatt tcaccgcaga aaatcagcca ttgtttttgg aaactccgag actcgcaacc 1080gaggtaaagg tgattgagtt cggtcaagtg gttgagcttg ttattcaagg gactagtttg 1140gttggtggtg gactcgatca tcctatgcat ctccatggtt ttagcttcta tgtggttgga 1200gtagggttcg ggaactataa cataagtgaa gaagatccgt cgtcgagata taatctctac 1260gatccaccgt ataaaaatac aatgaccgtg cctaggaatg gttggatcgc tatcagattc 1320gtagctgata atcccggggt ttggttcatg cactgtcact tggatagaca tcaaacgtgg 1380ggtatgaatg ttgtgttcat tgttaagaat ggtagagagc caaatcagca gattctgcct 1440ccaccagatg atttgccgcc ttgctatgaa taa 147332490PRTArabidopsis thaliana 32Met His Trp His Gly Val Glu Gln Pro Arg Asn Pro Trp Ser Asp Gly1 5 10 15Pro Glu Tyr Ile Thr Gln Cys Pro Ile Arg Pro Gly Ser Asp Phe Leu 20 25 30Tyr Lys Val Ile Phe Ser Ile Glu Asp Thr Thr Val Trp Trp His Ala 35 40 45His Ser Ser Trp Thr Arg Ala Thr Val His Gly Leu Ile Phe Val Tyr 50 55 60Pro Arg Pro Pro Gln Ile Leu Pro Phe Pro Lys Ala Asp His Glu Val65 70 75 80Pro Ile Ile Leu Gly Glu Trp Trp Lys Arg Asp Val Arg Glu Val Val 85 90 95Glu Glu Phe Val Arg Thr Gly Gly Ala Pro Asn Val Ser Asp Ala Leu 100 105 110Thr Ile Asn Gly His Pro Gly Phe Leu Tyr Pro Cys Ser Lys Ser Asp 115 120 125Thr Phe His Leu Thr Val Glu Lys Gly Lys Thr Tyr Arg Ile Arg Met 130 135 140Val Asn Ala Ala Met Asn Leu Pro Leu Phe Phe Ala Ile Ala Asn His145 150 155 160Ser Leu Thr Val Val Ser Ala Asp Gly His Tyr Ile Lys Pro Ile Lys 165 170 175Ala Thr Tyr Ile Thr Ile Ser Pro Gly Glu Thr Leu Asp Met Leu Leu 180 185 190His Ala Asp Gln Asp Pro Glu Arg Thr Tyr Tyr Met Ala Ala Arg Ala 195 200 205Tyr Gln Ser Gly Asn Ile Asp Phe Asn Asn Ser Thr Thr Ile Gly Ile 210 215 220Leu Ser Tyr Thr Ser Ser Cys Lys Ala Lys Thr Ser Ser Phe Ser Gly225 230 235 240Tyr Tyr Pro Thr Leu Pro Phe Tyr Asn Asp Thr Ser Ala Ala Phe Gly 245 250 255Phe Phe Thr Lys Ile Lys Cys Leu Phe Ser Gly Gln Val Pro Val Gln 260 265 270Ile Ser Arg Arg Ile Ile Thr Thr Val Ser Ile Asn Leu Arg Met Cys 275 280 285Pro Gln Asn Ser Cys Glu Gly Pro Asn Gly Ser Arg Leu Ala Ala Ser 290 295 300Met Asn Asn Ile Ser Phe Val Thr Pro Ser His Val Asp Ile Leu Lys305 310 315 320Ala Tyr Tyr Tyr His Ile Lys Gly Val Tyr Gly Thr Arg Phe Pro Glu 325 330 335Phe Pro Pro Leu Ile Phe Asn Phe Thr Ala Glu Asn Gln Pro Leu Phe 340 345 350Leu Glu Thr Pro Arg Leu Ala Thr Glu Val Lys Val Ile Glu Phe Gly 355 360 365Gln Val Val Glu Leu Val Ile Gln Gly Thr Ser Leu Val Gly Gly Gly 370 375 380Leu Asp His Pro Met His Leu His Gly Phe Ser Phe Tyr Val Val Gly385 390 395 400Val Gly Phe Gly Asn Tyr Asn Ile Ser Glu Glu Asp Pro Ser Ser Arg 405 410 415Tyr Asn Leu Tyr Asp Pro Pro Tyr Lys Asn Thr Met Thr Val Pro Arg 420 425 430Asn Gly Trp Ile Ala Ile Arg Phe Val Ala Asp Asn Pro Gly Val Trp 435 440 445Phe Met His Cys His Leu Asp Arg His Gln Thr Trp Gly Met Asn Val 450 455 460Val Phe Ile Val Lys Asn Gly Arg Glu Pro Asn Gln Gln Ile Leu Pro465 470 475 480Pro Pro Asp Asp Leu Pro Pro Cys Tyr Glu 485 490 331470DNAArabidopsis thaliana 33atgtctgctt ctgattcctc ttcctctctt cccgttactc ttgacaccat caaccccaag 60gttatcaaat gtgagtatgc tgtccgtgga gaaattgtca acattgctca gaaattgcaa 120gaagatttga agactaacaa ggacgcttat ccctttgatg agattatcta ctgtaatatc 180gggaatcctc aatctcttgg tcaacagcct ataacattct tcagagaggt tcttgcttta 240tgttcctaca cagccctgtt ggatgagagt gcaacacacg gtttgttcag ttctgattcg 300attgagcgtg cttggaagat tctggaccaa attcccggga gagcgactgg tgcttacagc 360cacagccagg gtatcaaggg gttacgtgat gcaattgctg atggaatcga agcccgtgat 420ggtttccctg ctgatcctaa tgatatattc atgacagatg gtgcaagtcc aggggttcat 480atgatgatgc aacttctcat aacttcagag aaagatggaa tcctttgtcc tattcctcag 540tatccattgt actcagcttc aattgccctt cacggtggaa ctttggttcc atactacctt 600gatgaagcat caggatgggg tcttgaaata tctgagctga agaaacaact tgaagatgct 660aggtcaaagg gcatcactgt gagagctttg gctgtcatta accctggaaa cccgacaggg 720caggttcttt cggaagaaaa ccagcgtgac gttgttaagt tctgcaagca agagggttta 780gttcttttag cagacgaggt ttatcaagag aatgtctatg tccctgacaa aaagttccat 840tccttcaaga aagtagcccg ctctatgggc tacggtgaga aggatcttgc cttagtctct 900ttccaatctg tctccaaagg gtactatgga gagtgtggga aaagaggtgg ttacatggag 960gttactggat tcacttctga tgtaagagag cagatataca aaatggcttc tgtgaatctt 1020tgttccaaca tctctggtca aattcttgct agcctcatca tgagcccacc caagcctggt 1080gacgactcct atgaatcata catagcagag aaggatggaa ttctctcatc tttggcaaga 1140cgtgcaaaga ctcttgaaga ggctctgaac aagctagagg gagttacatg caatagagca 1200gaaggagcta tgtatctatt cccttgcctt caccttccac aaaaggcaat tgcagctgct 1260gaggcggaaa agacagcacc agacaatttc tactgcaaac gccttctaaa agctactgga 1320atagtcgttg tccctggttc tggctttaga caggtacctg gaacatggca tttcaggtgc 1380actatacttc cccaagagga taagattcca gcgattgttg atcgtctaac tgcgttccac 1440cagagcttca tggacgagtt ccgcgactaa 147034489PRTArabidopsis thaliana 34Met Ser Ala Ser Asp Ser Ser Ser Ser Leu Pro Val Thr Leu Asp Thr1 5 10 15Ile Asn Pro Lys Val Ile Lys Cys Glu Tyr Ala Val Arg Gly Glu Ile 20 25 30Val Asn Ile Ala Gln Lys Leu Gln Glu Asp Leu Lys Thr Asn Lys Asp 35 40 45Ala Tyr Pro Phe Asp Glu Ile Ile Tyr Cys Asn Ile Gly Asn Pro Gln 50 55 60Ser Leu Gly Gln Gln Pro Ile Thr Phe Phe Arg Glu Val Leu Ala Leu65 70 75 80Cys Ser Tyr Thr Ala Leu Leu Asp Glu Ser Ala Thr His Gly Leu Phe 85 90 95Ser Ser Asp Ser Ile Glu Arg Ala Trp Lys Ile Leu Asp Gln Ile Pro 100 105 110Gly Arg Ala Thr Gly Ala Tyr Ser His Ser Gln Gly Ile Lys Gly Leu 115 120 125Arg Asp Ala Ile Ala Asp Gly Ile Glu Ala Arg Asp Gly Phe Pro Ala 130 135 140Asp Pro Asn Asp Ile Phe Met Thr Asp Gly Ala Ser Pro Gly Val His145 150 155 160Met Met Met Gln Leu Leu Ile Thr Ser Glu Lys Asp Gly Ile Leu Cys 165 170 175Pro Ile Pro Gln Tyr Pro Leu Tyr Ser Ala Ser Ile Ala Leu His Gly 180 185 190Gly Thr Leu Val Pro Tyr Tyr Leu Asp Glu Ala Ser Gly Trp Gly Leu 195 200 205Glu Ile Ser Glu Leu Lys Lys Gln Leu Glu Asp Ala Arg Ser Lys Gly 210 215 220Ile Thr Val Arg Ala Leu Ala Val Ile Asn Pro Gly Asn Pro Thr Gly225 230 235 240Gln Val Leu Ser Glu Glu Asn Gln Arg Asp Val Val Lys Phe Cys Lys 245 250 255Gln Glu Gly Leu Val Leu Leu Ala Asp Glu Val Tyr Gln Glu Asn Val 260 265 270Tyr Val Pro Asp Lys Lys Phe His Ser Phe Lys Lys Val Ala Arg Ser 275 280 285Met Gly Tyr Gly Glu Lys Asp Leu Ala Leu Val Ser Phe Gln Ser Val 290 295 300Ser Lys Gly Tyr Tyr Gly Glu Cys Gly Lys Arg Gly Gly Tyr Met Glu305 310 315 320Val Thr Gly Phe Thr Ser Asp Val Arg Glu Gln Ile Tyr Lys Met Ala 325 330 335Ser Val Asn Leu Cys Ser Asn Ile Ser Gly Gln Ile Leu Ala Ser Leu 340 345 350Ile Met Ser Pro Pro Lys Pro Gly Asp Asp Ser Tyr Glu Ser Tyr Ile 355 360 365Ala Glu Lys Asp Gly Ile Leu Ser Ser Leu Ala Arg Arg Ala Lys Thr 370 375 380Leu Glu Glu Ala Leu Asn Lys Leu Glu Gly Val Thr Cys Asn Arg Ala385 390 395 400Glu Gly Ala Met Tyr Leu Phe Pro Cys Leu His Leu Pro Gln Lys Ala 405 410 415Ile Ala Ala Ala Glu Ala Glu Lys Thr Ala Pro Asp Asn Phe Tyr Cys 420 425 430Lys Arg Leu Leu Lys Ala Thr Gly Ile Val Val Val Pro Gly Ser Gly 435 440 445Phe Arg Gln Val Pro Gly Thr Trp His Phe Arg Cys Thr Ile Leu Pro 450 455 460Gln Glu Asp Lys Ile Pro Ala Ile Val Asp Arg Leu Thr Ala Phe His465 470 475 480Gln Ser Phe Met Asp Glu Phe Arg Asp 48535519DNAArabidopsis thaliana 35atggcgttcc ctaaggtata cttcgacatg accatcgacg gccagcccgc gggaaggatc 60gtgatggagc tgtacaccga taagactccc aggactgccg agaatttcag agctctctgc 120accggagaga aaggtgttgg cggtaccgga aaaccccttc acttcaaggg atctaagttt 180caccgtgtga tccctaactt catgtgccag ggaggagatt tcaccgccgg gaacggaaca 240ggcggtgagt cgatctacgg gagcaagttc gaggacgaga atttcgagag gaagcacacc 300ggaccgggga tcctgtcgat ggcgaacgcc ggtgcaaaca cgaacggatc tcagttcttc 360atctgcaccg tgaagaccga ttggcttgat gggaagcacg tggtgtttgg gcaggtcgtg 420gaaggcttag acgtggtaaa ggccatcgag aaggttggat catcatctgg aaagccgacg 480aagcctgtgg ttgttgccga ttgtggtcag ctctcttag 51936172PRTArabidopsis thaliana 36Met Ala Phe Pro Lys Val Tyr Phe Asp Met Thr Ile Asp Gly Gln Pro1 5 10 15Ala Gly Arg Ile Val Met Glu Leu Tyr Thr Asp Lys Thr Pro Arg Thr 20 25 30Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly Val Gly Gly 35 40 45Thr Gly Lys Pro Leu His Phe Lys Gly Ser Lys Phe His Arg Val Ile 50 55 60Pro Asn Phe Met Cys Gln Gly Gly Asp Phe Thr Ala Gly Asn Gly Thr65 70 75 80Gly Gly Glu Ser Ile Tyr Gly Ser Lys Phe Glu Asp Glu Asn Phe Glu 85 90 95Arg Lys His Thr Gly Pro Gly Ile Leu Ser Met Ala Asn Ala Gly Ala 100 105 110Asn Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Val Lys Thr Asp Trp 115 120 125Leu Asp Gly Lys His Val Val Phe Gly Gln Val Val Glu Gly Leu Asp 130 135 140Val Val Lys Ala Ile Glu Lys Val Gly Ser Ser Ser Gly Lys Pro Thr145 150 155 160Lys Pro Val Val Val Ala Asp Cys Gly Gln Leu Ser 165 170371095DNAArabidopsis thaliana 37atgggtagtg caaaatcagc catgctgatc ctcttagtag caatggtcat cgcatcatgt 60gccacggcca ttgatatgtc cgtggtttcc tacgacgata acaaccgtct ccatagcgtt 120ttcgatgctg aggcctcgtt aatcttcgag tcatggatgg tcaaacatgg gaaagtgtac 180gggtccgttg ccgagaagga acggcgtttg acgatttttg aggacaacct ccgttttatc 240aataaccgga acgctgagaa tctcagttat cggcttggtt tgaccgggtt tgcggattta 300tctcttcatg agtataaaga agtttgccac ggggctgatc caagacctcc taggaaccac 360gtctttatga ctagtagcga ccgatacaag actagtgctg atgatgtgct tcctaagtca 420gttgattgga gaaacgaagg agcagtgact gaagtcaaag atcaaggcca ttgcaggagt 480tgttgggctt tctcgactgt gggagcagtg gaaggcttaa acaagatcgt gacgggagag 540ttagtcactt tgtctgagca agatttgatc aattgtaaca aagaaaacaa tggttgcgga 600ggaggcaaac tcgagactgc ctatgagttc atcatgaaaa atggtggtct tggtaccgac 660aacgattatc cttacaaagc tgttaacgga gtctgtgatg gccgcctcaa ggaaaacaac 720aagaatgtta tgattgatgg gtatgaaaat ttgcccgcaa acgacgaatc tgctctcatg 780aaagcggttg ctcaccagcc tgtgactgcc gttatcgatt ccagtagccg agagtttcag 840ctttatgaat cgggagtgtt tgatggctcg tgtggaacaa acctaaacca tggagttgtt 900gtggtcgggt atggaactga gaatggtcgt gactactggc tcgtgaaaaa ctctaggggc 960attacatggg gagaagctgg ctacatgaag atggctcgaa acattgccaa tccaagaggc 1020ttatgtggca ttgcaatgcg agcttcatac cctctaaaga actccttttc taccgataaa 1080agctccatcg cctaa 109538364PRTArabidopsis thaliana 38Met Gly Ser Ala Lys Ser Ala Met Leu Ile Leu Leu Val Ala Met Val1 5 10 15Ile Ala Ser Cys Ala Thr Ala Ile Asp Met Ser Val Val Ser Tyr Asp 20 25 30Asp Asn Asn Arg Leu His Ser Val Phe Asp Ala Glu Ala Ser Leu Ile 35 40 45Phe Glu Ser Trp Met Val Lys His Gly Lys Val Tyr Gly Ser Val Ala 50 55 60Glu Lys Glu Arg Arg Leu Thr Ile Phe Glu Asp Asn Leu Arg Phe Ile65 70 75 80Asn Asn Arg Asn Ala Glu Asn Leu Ser Tyr Arg Leu Gly Leu Thr Gly 85 90 95Phe Ala Asp Leu Ser Leu His Glu Tyr Lys Glu Val Cys His Gly Ala 100 105 110Asp Pro Arg Pro Pro Arg Asn His Val Phe Met Thr Ser Ser Asp Arg 115 120 125Tyr Lys Thr Ser Ala Asp Asp Val Leu Pro Lys Ser Val Asp Trp Arg 130 135 140Asn Glu Gly Ala Val Thr Glu Val Lys Asp Gln Gly His Cys Arg Ser145 150 155 160Cys Trp Ala Phe Ser Thr Val Gly Ala Val Glu Gly Leu Asn Lys Ile 165 170 175Val Thr Gly Glu Leu Val Thr Leu Ser Glu Gln Asp Leu Ile Asn Cys 180 185 190Asn Lys Glu Asn Asn Gly Cys Gly Gly Gly Lys Leu Glu Thr Ala Tyr 195 200 205Glu Phe Ile Met Lys Asn Gly Gly Leu Gly Thr Asp Asn Asp Tyr Pro 210 215 220Tyr Lys Ala Val Asn Gly Val Cys Asp Gly Arg Leu Lys Glu Asn Asn225 230 235 240Lys Asn Val Met Ile Asp Gly Tyr Glu Asn Leu Pro Ala Asn Asp Glu 245 250 255Ser Ala Leu Met Lys Ala Val Ala His Gln Pro Val Thr Ala Val Ile 260 265 270Asp Ser Ser Ser Arg Glu Phe Gln Leu Tyr Glu Ser Gly Val Phe Asp 275 280 285Gly Ser Cys Gly Thr Asn Leu Asn His Gly Val Val Val Val Gly Tyr 290 295 300Gly Thr Glu Asn Gly Arg Asp Tyr Trp Leu Val Lys Asn Ser Arg Gly305 310 315 320Ile Thr Trp Gly Glu Ala Gly Tyr Met Lys Met Ala Arg Asn Ile Ala 325 330 335Asn Pro Arg Gly Leu Cys Gly Ile Ala Met Arg Ala Ser Tyr Pro Leu 340 345 350Lys Asn Ser Phe Ser Thr Asp Lys Ser Ser Ile Ala 355 36039516DNAArabidopsis thaliana 39atgactaatc ccatgatcat ggttatgctg ttgttgtttc ttgtgatgtc gactagagca 60gacgaagagc tgattaagac agagtgtaat cacacagaat accaaaacgt atgcctcttc 120tgtcttgaag ccgatccaat ctccttcaat atcgaccgtg ctggacttgt caacatcatt 180atacactgtc tcggatctca acttgatgtt cttatcaaca ccgtcacgag tctaaagttg 240atgaaaggag agggtgaagc aaatgagaat gttctgaaag attgcgtcac aggctttgcg 300attgcacaat tacgacttca aggagccaac atcgatttga taacccttaa ttacgataaa 360gcgtacgaat tggtgaaaac tgcgttaaac tatcctcgga cttgcgaaga aaatctccaa 420aaactcaagt tcaaagattc atctgatgtt tatgacgata tcttggcata tagccaactc 480acctctgttg ctaagacgtt gatccaccgt ctctag 51640171PRTArabidopsis thaliana 40Met Thr Asn Pro Met Ile Met Val Met Leu Leu Leu Phe Leu Val Met1 5 10 15Ser Thr Arg Ala Asp Glu Glu Leu Ile Lys Thr Glu Cys Asn His Thr 20 25 30Glu Tyr Gln Asn Val Cys Leu Phe Cys Leu Glu Ala Asp Pro Ile Ser 35 40 45Phe Asn Ile Asp Arg Ala Gly Leu Val Asn Ile Ile Ile His Cys Leu 50 55 60Gly Ser Gln Leu Asp Val Leu Ile Asn Thr Val Thr Ser Leu Lys Leu65 70 75 80Met Lys Gly Glu Gly Glu Ala Asn Glu Asn Val Leu Lys Asp Cys Val 85 90 95Thr Gly Phe Ala Ile Ala Gln Leu Arg Leu Gln Gly Ala Asn Ile Asp 100 105 110Leu Ile Thr Leu Asn Tyr Asp Lys Ala Tyr Glu Leu Val Lys Thr Ala 115 120 125Leu Asn Tyr Pro Arg Thr Cys Glu Glu Asn Leu Gln Lys Leu Lys Phe 130 135 140Lys Asp Ser Ser Asp Val Tyr Asp Asp Ile Leu Ala Tyr Ser Gln Leu145 150 155 160Thr Ser Val Ala Lys Thr Leu Ile His Arg Leu 165 17041702DNAArabidopsis thaliana 41atggtttctt cttctttaac caagcttgtg ttctttggtt gtctcctcct gctcacattc 60acggacaacc ttgtggctgg aaaatctggc aaagtgaagc tcaatcttta ctacgaatca 120ctttgtcccg gttgtcagga attcatcgtc gatgacctag gtaaaatctt tgactacgat 180ctctacacaa tcactgatct caagctgttt
ccatttggta atgccgaact ctccgataat 240ctgactgtca cttgccagca tggtgaagag gaatgcaaac taaacgccct tgaagcttgc 300gcattaagaa cttggcccga tcagaaatca caatactcgt tcatacggtg cgtcgaaagc 360gatacgaaag gctgggaatc atgtgttaaa aactctggac gtgagaaagc aatcaatgat 420tgttacaatg gtgatctttc tagaaagctg atacttgggt acgcaaccaa aaccaagaat 480ttgaagccgc cacatgaata cgtaccatgg gtcacactca acggcaagcc actcgatgac 540agcgtacaaa gtacggatga tctcgtagct caaatctgca atgcatacaa aggaaagact 600actctcccaa aagtttgcaa ttcatccgcc tcaatgtcta agtcgcctga gaggaaatgg 660aagcttcaag tctcttatgc caataaagct accaattatt aa 70242233PRTArabidopsis thaliana 42Met Val Ser Ser Ser Leu Thr Lys Leu Val Phe Phe Gly Cys Leu Leu1 5 10 15Leu Leu Thr Phe Thr Asp Asn Leu Val Ala Gly Lys Ser Gly Lys Val 20 25 30Lys Leu Asn Leu Tyr Tyr Glu Ser Leu Cys Pro Gly Cys Gln Glu Phe 35 40 45Ile Val Asp Asp Leu Gly Lys Ile Phe Asp Tyr Asp Leu Tyr Thr Ile 50 55 60Thr Asp Leu Lys Leu Phe Pro Phe Gly Asn Ala Glu Leu Ser Asp Asn65 70 75 80Leu Thr Val Thr Cys Gln His Gly Glu Glu Glu Cys Lys Leu Asn Ala 85 90 95Leu Glu Ala Cys Ala Leu Arg Thr Trp Pro Asp Gln Lys Ser Gln Tyr 100 105 110Ser Phe Ile Arg Cys Val Glu Ser Asp Thr Lys Gly Trp Glu Ser Cys 115 120 125Val Lys Asn Ser Gly Arg Glu Lys Ala Ile Asn Asp Cys Tyr Asn Gly 130 135 140Asp Leu Ser Arg Lys Leu Ile Leu Gly Tyr Ala Thr Lys Thr Lys Asn145 150 155 160Leu Lys Pro Pro His Glu Tyr Val Pro Trp Val Thr Leu Asn Gly Lys 165 170 175Pro Leu Asp Asp Ser Val Gln Ser Thr Asp Asp Leu Val Ala Gln Ile 180 185 190Cys Asn Ala Tyr Lys Gly Lys Thr Thr Leu Pro Lys Val Cys Asn Ser 195 200 205Ser Ala Ser Met Ser Lys Ser Pro Glu Arg Lys Trp Lys Leu Gln Val 210 215 220Ser Tyr Ala Asn Lys Ala Thr Asn Tyr225 23043591DNAArabidopsis thaliana 43atggccggag ttttcaaaac ggttacgttt cttgttttgg ttttcgctgc cgttgttgtc 60ttcgcggagg actacgatgt tggtgatgat acggaatgga cgagacctat ggaccccgag 120ttctatacta cttgggctac cggtaaaact ttccgtgtag gcgacgagct cgaatttgat 180ttcgctgctg ggaggcatga tgtggcagtt gtatcagaag ctgcatttga aaactgtgag 240aaagagaaac ccattagcca catgaccgtt cctccggtca aaattatgct aaacaccact 300ggaccacaat actttatctg caccgtcggt gaccattgtc gttttggtca aaaactttcc 360atcactgtag ttgctgctgg tgcaactgga ggtgctactc ctggtgccgg tgctacccca 420gcacctggat caaccccaag tactggagga accactcctc ccactgcggg tgggaccaca 480acaccttcag gctctagcgg aaccactact ccagctggaa atgccgcttc ctcattaggt 540ggtgctactt ttctggtcgc ttttgtttct gctgttgttg ctctcttttg a 59144196PRTArabidopsis thaliana 44Met Ala Gly Val Phe Lys Thr Val Thr Phe Leu Val Leu Val Phe Ala1 5 10 15Ala Val Val Val Phe Ala Glu Asp Tyr Asp Val Gly Asp Asp Thr Glu 20 25 30Trp Thr Arg Pro Met Asp Pro Glu Phe Tyr Thr Thr Trp Ala Thr Gly 35 40 45Lys Thr Phe Arg Val Gly Asp Glu Leu Glu Phe Asp Phe Ala Ala Gly 50 55 60Arg His Asp Val Ala Val Val Ser Glu Ala Ala Phe Glu Asn Cys Glu65 70 75 80Lys Glu Lys Pro Ile Ser His Met Thr Val Pro Pro Val Lys Ile Met 85 90 95Leu Asn Thr Thr Gly Pro Gln Tyr Phe Ile Cys Thr Val Gly Asp His 100 105 110Cys Arg Phe Gly Gln Lys Leu Ser Ile Thr Val Val Ala Ala Gly Ala 115 120 125Thr Gly Gly Ala Thr Pro Gly Ala Gly Ala Thr Pro Ala Pro Gly Ser 130 135 140Thr Pro Ser Thr Gly Gly Thr Thr Pro Pro Thr Ala Gly Gly Thr Thr145 150 155 160Thr Pro Ser Gly Ser Ser Gly Thr Thr Thr Pro Ala Gly Asn Ala Ala 165 170 175Ser Ser Leu Gly Gly Ala Thr Phe Leu Val Ala Phe Val Ser Ala Val 180 185 190Val Ala Leu Phe 19545459DNAArabidopsis thaliana 45atggcgtcaa agcaactgag cagagaagag cttgatgaga aggcgaagca aggagagacc 60gtcgtcccag gtggcaccgg tggccacagc ctcgaagctc aagagcatct tgctgaagga 120aggagcaagg gagggcagac gaggaaggag cagctaggac atgaaggtta tcaggagatc 180ggtcacaaag gtggagaggc gaggaaggag cagttagggc acgagggtta tcaggagatg 240ggtcacaaag gtggagaggc gaggaaggag cagctagggc acgagggtta tcaggagatg 300ggacacaaag gaggagaggc gaggaaggag cagctagggc acgagggtta taaggagatg 360ggacgtaaag gaggactcag tacgatggaa aaatctggtg gagagcgtgc ggaggaagaa 420gggattgaga tcgatgagtc aaagttcacc aacaagtga 45946152PRTArabidopsis thaliana 46Met Ala Ser Lys Gln Leu Ser Arg Glu Glu Leu Asp Glu Lys Ala Lys1 5 10 15Gln Gly Glu Thr Val Val Pro Gly Gly Thr Gly Gly His Ser Leu Glu 20 25 30Ala Gln Glu His Leu Ala Glu Gly Arg Ser Lys Gly Gly Gln Thr Arg 35 40 45Lys Glu Gln Leu Gly His Glu Gly Tyr Gln Glu Ile Gly His Lys Gly 50 55 60Gly Glu Ala Arg Lys Glu Gln Leu Gly His Glu Gly Tyr Gln Glu Met65 70 75 80Gly His Lys Gly Gly Glu Ala Arg Lys Glu Gln Leu Gly His Glu Gly 85 90 95Tyr Gln Glu Met Gly His Lys Gly Gly Glu Ala Arg Lys Glu Gln Leu 100 105 110Gly His Glu Gly Tyr Lys Glu Met Gly Arg Lys Gly Gly Leu Ser Thr 115 120 125Met Glu Lys Ser Gly Gly Glu Arg Ala Glu Glu Glu Gly Ile Glu Ile 130 135 140Asp Glu Ser Lys Phe Thr Asn Lys145 150471191DNAArabidopsis thaliana 47atggcttcgg ttactttctc tgtccccaag ggtttcactg aattctcagg attgcgaagc 60tcctctgctt ctcttccctt cggcaagaaa ctttcttccg atgagttcgt ttccatcgtc 120tccttccaga cttctgcaat gggaagcagt ggtggataca ggaaaggtgt gactgaggcc 180aagcttaagg tggccattaa tggattcggt aggatcggga ggaacttcct gagatgttgg 240catggtcgca aggactctcc tcttgatatc attgccatta atgacactgg tggcgtcaag 300caggcttcgc atttacttaa atacgactct actctcggaa tctttgatgc tgatgtcaaa 360ccttctggag agactgcaat ctctgttgat ggaaagatca tccaagttgt ctctaaccga 420aacccgtctc ttctcccttg gaaggagcta ggaattgaca ttgtcatcga aggaaccgga 480gtgtttgtgg atagagaagg tgcagggaaa cacattgaag ctggtgccaa gaaggttatc 540attactgctc caggcaaagg agatattcca acttatgtcg ttggtgtcaa tgcagatgct 600tacagtcatg atgaacctat catcagcaat gcatcttgca ctaccaactg tcttgctccc 660tttgtcaaag ttcttgacca gaaattcggt atcataaagg gtacaatgac gactactcac 720tcttacaccg gtgaccagag gttgctagac gcgagtcacc gtgatctaag gagagcaaga 780gcagctgctt tgaacattgt tcctacttct acaggagcag ctaaagctgt ggctcttgtg 840ctccctaacc tcaaaggaaa actcaacggg atcgctctcc gtgtaccaac accaaacgta 900tcagtggttg atctcgttgt gcaggtctca aagaagacat ttgctgagga agtcaacgct 960gctttcagag attctgcaga gaaagagctt aaaggtatac tcgatgtctg cgatgagcca 1020ctagtgtccg ttgatttcag atgctcagat ttttcaacga ccattgattc atcactcact 1080atggttatgg gagatgatat ggttaaggtg attgcttggt atgataatga atggggttac 1140tcacagagag ttgttgactt ggctgacatt gttgccaaca actggaagtg a 119148396PRTArabidopsis thaliana 48Met Ala Ser Val Thr Phe Ser Val Pro Lys Gly Phe Thr Glu Phe Ser1 5 10 15Gly Leu Arg Ser Ser Ser Ala Ser Leu Pro Phe Gly Lys Lys Leu Ser 20 25 30Ser Asp Glu Phe Val Ser Ile Val Ser Phe Gln Thr Ser Ala Met Gly 35 40 45Ser Ser Gly Gly Tyr Arg Lys Gly Val Thr Glu Ala Lys Leu Lys Val 50 55 60Ala Ile Asn Gly Phe Gly Arg Ile Gly Arg Asn Phe Leu Arg Cys Trp65 70 75 80His Gly Arg Lys Asp Ser Pro Leu Asp Ile Ile Ala Ile Asn Asp Thr 85 90 95Gly Gly Val Lys Gln Ala Ser His Leu Leu Lys Tyr Asp Ser Thr Leu 100 105 110Gly Ile Phe Asp Ala Asp Val Lys Pro Ser Gly Glu Thr Ala Ile Ser 115 120 125Val Asp Gly Lys Ile Ile Gln Val Val Ser Asn Arg Asn Pro Ser Leu 130 135 140Leu Pro Trp Lys Glu Leu Gly Ile Asp Ile Val Ile Glu Gly Thr Gly145 150 155 160Val Phe Val Asp Arg Glu Gly Ala Gly Lys His Ile Glu Ala Gly Ala 165 170 175Lys Lys Val Ile Ile Thr Ala Pro Gly Lys Gly Asp Ile Pro Thr Tyr 180 185 190Val Val Gly Val Asn Ala Asp Ala Tyr Ser His Asp Glu Pro Ile Ile 195 200 205Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Phe Val Lys Val 210 215 220Leu Asp Gln Lys Phe Gly Ile Ile Lys Gly Thr Met Thr Thr Thr His225 230 235 240Ser Tyr Thr Gly Asp Gln Arg Leu Leu Asp Ala Ser His Arg Asp Leu 245 250 255Arg Arg Ala Arg Ala Ala Ala Leu Asn Ile Val Pro Thr Ser Thr Gly 260 265 270Ala Ala Lys Ala Val Ala Leu Val Leu Pro Asn Leu Lys Gly Lys Leu 275 280 285Asn Gly Ile Ala Leu Arg Val Pro Thr Pro Asn Val Ser Val Val Asp 290 295 300Leu Val Val Gln Val Ser Lys Lys Thr Phe Ala Glu Glu Val Asn Ala305 310 315 320Ala Phe Arg Asp Ser Ala Glu Lys Glu Leu Lys Gly Ile Leu Asp Val 325 330 335Cys Asp Glu Pro Leu Val Ser Val Asp Phe Arg Cys Ser Asp Phe Ser 340 345 350Thr Thr Ile Asp Ser Ser Leu Thr Met Val Met Gly Asp Asp Met Val 355 360 365Lys Val Ile Ala Trp Tyr Asp Asn Glu Trp Gly Tyr Ser Gln Arg Val 370 375 380Val Asp Leu Ala Asp Ile Val Ala Asn Asn Trp Lys385 390 395491569DNAArabidopsis thaliana 49atggcaaaag aaaatggatt tataggatca atcgatcaag gaaccaccag caccagattc 60atcatttacg accacgatgc tcgtcctgtt gcttctcatc aagtcgagtt cactcagttc 120tatcccgaag ctggatgggt ggaacacgat ccaatggaga tactggaaag tgtgaaagtg 180tgcattgcaa aggctctcga caaagccact gccgatggac acaacgtcga cggtggcttg 240aaggccattg ggcttacaga tcagagagag actactgttg tttggagcaa atccactggc 300cttcctctcc acaaggctat tgtctggatg gatgctcgta ccagctccat ctgcaggaga 360ctagagaaag aactctcggg aggaagatcc cattttgtgg agtcttgcgg cttgccaata 420agcacatact tctctgccat gaagctgctt tggctcatgg agaatgtgga tgatgtcaaa 480gacgctatca agaaagggga tgccatcttt ggcactatcg acacatggtt gatctggaac 540atgactggcg gtatcaatgg cggccttcat gtcactgatg tcaccaatgc ttcacggaca 600atgctcatga acctcaaaac cttgagctgg gaccaggaca ctttgaagac acttggcata 660ccggctgaaa tcttgcccaa gattgtcagc aattcagaag tgattggaga gatctgcaaa 720ggctggccta ttcccggtat caagattgct ggatgtcttg gtgatcagca tgctgcgatg 780ttggggcaag cttgcagaaa aggcgaggcg aagagtactt atggcacagg tgctttcatt 840cttctcaaca ccggagaagt gccaatcaaa tcaggtcatg gtcttctgac cacgttggcc 900tacaagctcg gtcctcaagc acagacaaac tatgcattgg agggttcgat tgccatagca 960ggagctgctg ttcagtggct tagagacagc cttgggataa tcaaaagcgc ctctgagatc 1020gaagatttgg cagcaatggt agattctaca ggaggagtgt actttgtgcc agcgttcaac 1080ggcttgtttg ctccttggtg gagagaagac gcacgtggtg tgtgcattgg aatcacgagg 1140ttcaccaaca agtctcacat tgctcgggct gtgctggaga gcatgtgttt ccaggtgaaa 1200gacgtccttg actccatgaa caaagatgca ggtgaaaagg gttcccttaa taacgggaaa 1260ggggagttct tgctcagagt tgatggtggt gccacagcta acaaccttct gatgcagatt 1320caggctgatc tgatgggaag tccggtggtg aggccagtgg acatagagac aacagcatta 1380ggagcagcct atgcagctgg attagctgtg ggattctgga aggaagcaga catattcgag 1440tcgggagaga aggcgaagaa ctccaaagtt ttcagacccg ctatggaaga aggaatcagg 1500aagaagaaag tggcgtcttg gtgcaaagcg gtggaaagaa catttgatct cgctgacctc 1560tctatctaa 156950522PRTArabidopsis thaliana 50Met Ala Lys Glu Asn Gly Phe Ile Gly Ser Ile Asp Gln Gly Thr Thr1 5 10 15Ser Thr Arg Phe Ile Ile Tyr Asp His Asp Ala Arg Pro Val Ala Ser 20 25 30His Gln Val Glu Phe Thr Gln Phe Tyr Pro Glu Ala Gly Trp Val Glu 35 40 45His Asp Pro Met Glu Ile Leu Glu Ser Val Lys Val Cys Ile Ala Lys 50 55 60Ala Leu Asp Lys Ala Thr Ala Asp Gly His Asn Val Asp Gly Gly Leu65 70 75 80Lys Ala Ile Gly Leu Thr Asp Gln Arg Glu Thr Thr Val Val Trp Ser 85 90 95Lys Ser Thr Gly Leu Pro Leu His Lys Ala Ile Val Trp Met Asp Ala 100 105 110Arg Thr Ser Ser Ile Cys Arg Arg Leu Glu Lys Glu Leu Ser Gly Gly 115 120 125Arg Ser His Phe Val Glu Ser Cys Gly Leu Pro Ile Ser Thr Tyr Phe 130 135 140Ser Ala Met Lys Leu Leu Trp Leu Met Glu Asn Val Asp Asp Val Lys145 150 155 160Asp Ala Ile Lys Lys Gly Asp Ala Ile Phe Gly Thr Ile Asp Thr Trp 165 170 175Leu Ile Trp Asn Met Thr Gly Gly Ile Asn Gly Gly Leu His Val Thr 180 185 190Asp Val Thr Asn Ala Ser Arg Thr Met Leu Met Asn Leu Lys Thr Leu 195 200 205Ser Trp Asp Gln Asp Thr Leu Lys Thr Leu Gly Ile Pro Ala Glu Ile 210 215 220Leu Pro Lys Ile Val Ser Asn Ser Glu Val Ile Gly Glu Ile Cys Lys225 230 235 240Gly Trp Pro Ile Pro Gly Ile Lys Ile Ala Gly Cys Leu Gly Asp Gln 245 250 255His Ala Ala Met Leu Gly Gln Ala Cys Arg Lys Gly Glu Ala Lys Ser 260 265 270Thr Tyr Gly Thr Gly Ala Phe Ile Leu Leu Asn Thr Gly Glu Val Pro 275 280 285Ile Lys Ser Gly His Gly Leu Leu Thr Thr Leu Ala Tyr Lys Leu Gly 290 295 300Pro Gln Ala Gln Thr Asn Tyr Ala Leu Glu Gly Ser Ile Ala Ile Ala305 310 315 320Gly Ala Ala Val Gln Trp Leu Arg Asp Ser Leu Gly Ile Ile Lys Ser 325 330 335Ala Ser Glu Ile Glu Asp Leu Ala Ala Met Val Asp Ser Thr Gly Gly 340 345 350Val Tyr Phe Val Pro Ala Phe Asn Gly Leu Phe Ala Pro Trp Trp Arg 355 360 365Glu Asp Ala Arg Gly Val Cys Ile Gly Ile Thr Arg Phe Thr Asn Lys 370 375 380Ser His Ile Ala Arg Ala Val Leu Glu Ser Met Cys Phe Gln Val Lys385 390 395 400Asp Val Leu Asp Ser Met Asn Lys Asp Ala Gly Glu Lys Gly Ser Leu 405 410 415Asn Asn Gly Lys Gly Glu Phe Leu Leu Arg Val Asp Gly Gly Ala Thr 420 425 430Ala Asn Asn Leu Leu Met Gln Ile Gln Ala Asp Leu Met Gly Ser Pro 435 440 445Val Val Arg Pro Val Asp Ile Glu Thr Thr Ala Leu Gly Ala Ala Tyr 450 455 460Ala Ala Gly Leu Ala Val Gly Phe Trp Lys Glu Ala Asp Ile Phe Glu465 470 475 480Ser Gly Glu Lys Ala Lys Asn Ser Lys Val Phe Arg Pro Ala Met Glu 485 490 495Glu Gly Ile Arg Lys Lys Lys Val Ala Ser Trp Cys Lys Ala Val Glu 500 505 510Arg Thr Phe Asp Leu Ala Asp Leu Ser Ile 515 52051741DNAArabidopsis thaliana 51atggcggcga aaattcccgg agtgatcgct ttgttcgacg tcgacggtac tctcacagct 60ccaaggaagg aagctactcc agaattgctc gattttatcc gagaattgcg aaaggtcgtc 120actattggag tcgtcggtgg atctgatcta agtaagatat ctgagcagct tggcaaaaca 180gtcacaaacg actatgatta ttgtttctct gagaatggtc ttgtcgccca taaagatggg 240aaatccattg gaattcagag cctgaagctg caccttggag acgataaact caaggagttg 300ataaatttca cgctgcacta cattgcagac ctggatattc caattaagag gggaacattt 360attgaattcc gaaatggaat gctcaatgta tcacccattg gtcgcaactg cagccaagaa 420gaaagagatg aatttgagag atatgataag gttcaaaaca tccgaccaaa gatggtagct 480gaacttcgtg agcggtttgc acatcttaac cttactttct caattggggg acagataagc 540ttcgatgtct tccctaaagg ttgggataag acttactgct tgcaatacct cgaggacttc 600agtgaaatcc atttcttcgg tgacaagacc tatgagggtg gaaatgacta tgaaatctat 660gaatcaccaa aaacaattgg ccattcagtt acgagtccag atgacacagt ggcaaaatgc 720aaggctctgt tcatgtcttg a 74152246PRTArabidopsis thaliana 52Met Ala Ala Lys Ile Pro Gly Val Ile Ala Leu Phe Asp Val Asp Gly1 5 10 15Thr Leu Thr Ala Pro Arg Lys Glu Ala Thr Pro Glu Leu Leu Asp Phe 20 25 30Ile Arg Glu Leu Arg Lys Val Val Thr Ile Gly Val Val Gly Gly Ser 35 40 45Asp Leu Ser Lys Ile Ser Glu Gln Leu Gly Lys Thr Val Thr Asn Asp 50 55 60Tyr Asp Tyr Cys Phe Ser Glu Asn Gly Leu Val Ala His Lys Asp Gly65 70 75
80Lys Ser Ile Gly Ile Gln Ser Leu Lys Leu His Leu Gly Asp Asp Lys 85 90 95Leu Lys Glu Leu Ile Asn Phe Thr Leu His Tyr Ile Ala Asp Leu Asp 100 105 110Ile Pro Ile Lys Arg Gly Thr Phe Ile Glu Phe Arg Asn Gly Met Leu 115 120 125Asn Val Ser Pro Ile Gly Arg Asn Cys Ser Gln Glu Glu Arg Asp Glu 130 135 140Phe Glu Arg Tyr Asp Lys Val Gln Asn Ile Arg Pro Lys Met Val Ala145 150 155 160Glu Leu Arg Glu Arg Phe Ala His Leu Asn Leu Thr Phe Ser Ile Gly 165 170 175Gly Gln Ile Ser Phe Asp Val Phe Pro Lys Gly Trp Asp Lys Thr Tyr 180 185 190Cys Leu Gln Tyr Leu Glu Asp Phe Ser Glu Ile His Phe Phe Gly Asp 195 200 205Lys Thr Tyr Glu Gly Gly Asn Asp Tyr Glu Ile Tyr Glu Ser Pro Lys 210 215 220Thr Ile Gly His Ser Val Thr Ser Pro Asp Asp Thr Val Ala Lys Cys225 230 235 240Lys Ala Leu Phe Met Ser 245531170DNAArabidopsis thaliana 53 atgggttaca taggagctca tggtgtagca gctcttcata ggtacaaata cagtggagtg 60gatcactctt atcttgccaa atacgtcctc caaccttttt ggactcgatt tgtcaaagtc 120ttccctctat ggatgccacc aaacatgata acgcttatgg ggtttatgtt tctagtcact 180tcctccctgc taggctatat atattcacct cagttggatt ctcctcctcc acgatgggtt 240cacttcgcac atggtttact tctcttcttg tatcagacat ttgatgcggt tgatgggaag 300caagcaagaa ggacaaattc ctctagcccc ctaggagagc tcttcgatca tggttgtgac 360gcgcttgctt gtgcgtttga agccatggca tttggaagca ctgcaatgtg tggaagagat 420actttctggt tctgggttat ttcagctatt ccattttatg gagctacatg ggaacactat 480ttcaccaaca cacttattct tccggttatc aatgggccta cagaggggct tgcacttata 540tttgtcagcc acttcttcac agccatcgtc ggtgctgaat ggtgggctca gcagttaggg 600cagtcaatac cattgtttag ttgggtgcca tttgtgaatg agattcaaac ttctagagca 660gtgctataca tgatgatcgc ttttgctgtt ataccaaccg ttgcattcaa tgtaacaaat 720gtctacaaag tcgttcgatc aagaaacggg agcatggtgt tagcgttagc tatgctgtat 780cccttcgttg tcttacttgg aggagttttg atatgggatt acttgtctcc aatcaatctc 840atagcaacat atcctcactt agttgtactc ggaactggac ttgcatttgg atttttagtg 900ggaagaatga ttcttgctca cttgtgtgat gagcctaaag gactaaaaac aaacatgtgc 960atgtcactac tctatcttcc ctttgcactt gcaaatgcgc taaccgcaag attgaatgct 1020ggggttcctc tagtcgacga attatgggtt cttcttggct actgcatatt cacagtgtca 1080ttatacttgc actttgcaac atcagtcatc catgagatca ctgaggccct tggaatctac 1140tgctttagga tcacgcgtaa agaagcttga 117054389PRTArabidopsis thaliana 54Met Gly Tyr Ile Gly Ala His Gly Val Ala Ala Leu His Arg Tyr Lys1 5 10 15Tyr Ser Gly Val Asp His Ser Tyr Leu Ala Lys Tyr Val Leu Gln Pro 20 25 30Phe Trp Thr Arg Phe Val Lys Val Phe Pro Leu Trp Met Pro Pro Asn 35 40 45Met Ile Thr Leu Met Gly Phe Met Phe Leu Val Thr Ser Ser Leu Leu 50 55 60Gly Tyr Ile Tyr Ser Pro Gln Leu Asp Ser Pro Pro Pro Arg Trp Val65 70 75 80His Phe Ala His Gly Leu Leu Leu Phe Leu Tyr Gln Thr Phe Asp Ala 85 90 95Val Asp Gly Lys Gln Ala Arg Arg Thr Asn Ser Ser Ser Pro Leu Gly 100 105 110Glu Leu Phe Asp His Gly Cys Asp Ala Leu Ala Cys Ala Phe Glu Ala 115 120 125Met Ala Phe Gly Ser Thr Ala Met Cys Gly Arg Asp Thr Phe Trp Phe 130 135 140Trp Val Ile Ser Ala Ile Pro Phe Tyr Gly Ala Thr Trp Glu His Tyr145 150 155 160Phe Thr Asn Thr Leu Ile Leu Pro Val Ile Asn Gly Pro Thr Glu Gly 165 170 175Leu Ala Leu Ile Phe Val Ser His Phe Phe Thr Ala Ile Val Gly Ala 180 185 190Glu Trp Trp Ala Gln Gln Leu Gly Gln Ser Ile Pro Leu Phe Ser Trp 195 200 205Val Pro Phe Val Asn Glu Ile Gln Thr Ser Arg Ala Val Leu Tyr Met 210 215 220Met Ile Ala Phe Ala Val Ile Pro Thr Val Ala Phe Asn Val Thr Asn225 230 235 240Val Tyr Lys Val Val Arg Ser Arg Asn Gly Ser Met Val Leu Ala Leu 245 250 255Ala Met Leu Tyr Pro Phe Val Val Leu Leu Gly Gly Val Leu Ile Trp 260 265 270Asp Tyr Leu Ser Pro Ile Asn Leu Ile Ala Thr Tyr Pro His Leu Val 275 280 285Val Leu Gly Thr Gly Leu Ala Phe Gly Phe Leu Val Gly Arg Met Ile 290 295 300Leu Ala His Leu Cys Asp Glu Pro Lys Gly Leu Lys Thr Asn Met Cys305 310 315 320Met Ser Leu Leu Tyr Leu Pro Phe Ala Leu Ala Asn Ala Leu Thr Ala 325 330 335Arg Leu Asn Ala Gly Val Pro Leu Val Asp Glu Leu Trp Val Leu Leu 340 345 350Gly Tyr Cys Ile Phe Thr Val Ser Leu Tyr Leu His Phe Ala Thr Ser 355 360 365Val Ile His Glu Ile Thr Glu Ala Leu Gly Ile Tyr Cys Phe Arg Ile 370 375 380Thr Arg Lys Glu Ala385552019DNAArabidopsis thaliana 55atgtctcctt ctcactccat caattccaac agtgacggca acgcctcctg ttcaacctgc 60agcacctgct tcgatactac aacctccaca acagatgaag agtatgatgc tttgattaag 120actaagactt ggagtttagt gcctaaacct gcaggtacaa acattatcaa ttcgatttgg 180ctttataaac acaagtacaa tgctgatggt tctttgtcaa gatacaaatc gagactcgtt 240gctaatggaa aatcacaaga acacggagta gatttttatg agacgttcag tccggttgta 300aagcctgcaa caatccgtgc ggttctaaac tttgcggtgg aacgcgactc gtcggtacat 360caattagatg ttcaaaacgc gtttcttcat ggcaagctag aagaaactgt ttacatgtat 420gagccacctg gatttgtcga taacaagaat ccaggatatg tttgcaagct gaataaggcg 480ttgtatggtt ttaaacaagc accacgtgct tggaatgctc gatttgcaag ctatgtcaaa 540atgatgggtt tcaaacagag taagtgtgat gcttcacttt tcgtatacaa gcaagggaat 600gacatggctt acttgctgct ctacgttgat gacattatgc taactgcttc ttccccaagt 660caagaaaaat atgcgaagaa tattattaac atagttgaga tgcaaaactg gaagcctagt 720ctcacatcgg ttgatcttgc gtgtaagctt gaagaaagtg ttggtccaaa aatacaagac 780ctgacgttat atcgaagttt ggttggggca cttcagtact taacaataac gagaccgaac 840atatcttatg cagttcaaca aggtctcagt attacaaagt ccccatcaac caaactagtt 900gccgactcgg acgcagactg ggttgggtgt ccgaatacta gaagatcgac ttcggggtat 960tgtgtttttc ttagggatac actcatctca tggtcgtcta agcgacaagg ttcagtgtca 1020cgttcaagcg cagaggctga gtacaaagcc gtagcaaatg cagtggtagc aacatgcgaa 1080gttcgagtca ttcatatccc tgcttctcat caatatgctg acattttcac aaagggtctt 1140actacttcac tgttcaatcg attcaagtcc agtcttggcg tcattcaacg accgacgaaa 1200agactgcggg aaggtgattt gttgttcaac agaaaagatg agattcgtac gttcgaggtt 1260gtctctcgtt atggtcacgg tcctaagctt cttggccggt tttctggcgg tcgaatcgag 1320gagtttatta atgcccggac gttatcagca gcaaatctac gtgacgcgga agtacctact 1380cgtgttgcgg ctaagctaag agagtttcat ggtatcaaca tccctggtga tagaaatgtg 1440ctcatttggg ataggatgag gaattggctt agacaagcca aaagtctgtg tacacctgaa 1500gatttagcag agtttggtct agacaagatt gaagatgaga tatacttgct ggaacatgag 1560ctgcaggata agtgtaagca gcaggagata gggttttgtc acaatgattt gcaatatggt 1620aacattatga ttgatgaaga taccaatgcc attactatca ttgactacga gtacgctagt 1680tataacccag ttgcatacga cattgcaaat cacttctgcg aaatggcagc aaattacaaa 1740gttgcaggag aagaggaacg aaggaggttc atccataact acctcagctc ttcaggcgaa 1800gaaccaaaag aagatgacat aaaacagctc ttgaaggatg ctgagaagta cacattagca 1860agccatctgt tttggggctt atggggaatc atctctggat atgtaaacaa gatcgacttc 1920gattacgccg agtactcaag acagagattc aaacaatact ggcttcgaaa acccgagctc 1980ttactcttct cccaaatgta tatttcaaac accaaatga 201956672PRTArabidopsis thaliana 56Met Ser Pro Ser His Ser Ile Asn Ser Asn Ser Asp Gly Asn Ala Ser1 5 10 15Cys Ser Thr Cys Ser Thr Cys Phe Asp Thr Thr Thr Ser Thr Thr Asp 20 25 30Glu Glu Tyr Asp Ala Leu Ile Lys Thr Lys Thr Trp Ser Leu Val Pro 35 40 45Lys Pro Ala Gly Thr Asn Ile Ile Asn Ser Ile Trp Leu Tyr Lys His 50 55 60Lys Tyr Asn Ala Asp Gly Ser Leu Ser Arg Tyr Lys Ser Arg Leu Val65 70 75 80Ala Asn Gly Lys Ser Gln Glu His Gly Val Asp Phe Tyr Glu Thr Phe 85 90 95Ser Pro Val Val Lys Pro Ala Thr Ile Arg Ala Val Leu Asn Phe Ala 100 105 110Val Glu Arg Asp Ser Ser Val His Gln Leu Asp Val Gln Asn Ala Phe 115 120 125Leu His Gly Lys Leu Glu Glu Thr Val Tyr Met Tyr Glu Pro Pro Gly 130 135 140Phe Val Asp Asn Lys Asn Pro Gly Tyr Val Cys Lys Leu Asn Lys Ala145 150 155 160Leu Tyr Gly Phe Lys Gln Ala Pro Arg Ala Trp Asn Ala Arg Phe Ala 165 170 175Ser Tyr Val Lys Met Met Gly Phe Lys Gln Ser Lys Cys Asp Ala Ser 180 185 190Leu Phe Val Tyr Lys Gln Gly Asn Asp Met Ala Tyr Leu Leu Leu Tyr 195 200 205Val Asp Asp Ile Met Leu Thr Ala Ser Ser Pro Ser Gln Glu Lys Tyr 210 215 220Ala Lys Asn Ile Ile Asn Ile Val Glu Met Gln Asn Trp Lys Pro Ser225 230 235 240Leu Thr Ser Val Asp Leu Ala Cys Lys Leu Glu Glu Ser Val Gly Pro 245 250 255Lys Ile Gln Asp Leu Thr Leu Tyr Arg Ser Leu Val Gly Ala Leu Gln 260 265 270Tyr Leu Thr Ile Thr Arg Pro Asn Ile Ser Tyr Ala Val Gln Gln Gly 275 280 285Leu Ser Ile Thr Lys Ser Pro Ser Thr Lys Leu Val Ala Asp Ser Asp 290 295 300Ala Asp Trp Val Gly Cys Pro Asn Thr Arg Arg Ser Thr Ser Gly Tyr305 310 315 320Cys Val Phe Leu Arg Asp Thr Leu Ile Ser Trp Ser Ser Lys Arg Gln 325 330 335Gly Ser Val Ser Arg Ser Ser Ala Glu Ala Glu Tyr Lys Ala Val Ala 340 345 350Asn Ala Val Val Ala Thr Cys Glu Val Arg Val Ile His Ile Pro Ala 355 360 365Ser His Gln Tyr Ala Asp Ile Phe Thr Lys Gly Leu Thr Thr Ser Leu 370 375 380Phe Asn Arg Phe Lys Ser Ser Leu Gly Val Ile Gln Arg Pro Thr Lys385 390 395 400Arg Leu Arg Glu Gly Asp Leu Leu Phe Asn Arg Lys Asp Glu Ile Arg 405 410 415Thr Phe Glu Val Val Ser Arg Tyr Gly His Gly Pro Lys Leu Leu Gly 420 425 430Arg Phe Ser Gly Gly Arg Ile Glu Glu Phe Ile Asn Ala Arg Thr Leu 435 440 445Ser Ala Ala Asn Leu Arg Asp Ala Glu Val Pro Thr Arg Val Ala Ala 450 455 460Lys Leu Arg Glu Phe His Gly Ile Asn Ile Pro Gly Asp Arg Asn Val465 470 475 480Leu Ile Trp Asp Arg Met Arg Asn Trp Leu Arg Gln Ala Lys Ser Leu 485 490 495Cys Thr Pro Glu Asp Leu Ala Glu Phe Gly Leu Asp Lys Ile Glu Asp 500 505 510Glu Ile Tyr Leu Leu Glu His Glu Leu Gln Asp Lys Cys Lys Gln Gln 515 520 525Glu Ile Gly Phe Cys His Asn Asp Leu Gln Tyr Gly Asn Ile Met Ile 530 535 540Asp Glu Asp Thr Asn Ala Ile Thr Ile Ile Asp Tyr Glu Tyr Ala Ser545 550 555 560Tyr Asn Pro Val Ala Tyr Asp Ile Ala Asn His Phe Cys Glu Met Ala 565 570 575Ala Asn Tyr Lys Val Ala Gly Glu Glu Glu Arg Arg Arg Phe Ile His 580 585 590Asn Tyr Leu Ser Ser Ser Gly Glu Glu Pro Lys Glu Asp Asp Ile Lys 595 600 605Gln Leu Leu Lys Asp Ala Glu Lys Tyr Thr Leu Ala Ser His Leu Phe 610 615 620Trp Gly Leu Trp Gly Ile Ile Ser Gly Tyr Val Asn Lys Ile Asp Phe625 630 635 640Asp Tyr Ala Glu Tyr Ser Arg Gln Arg Phe Lys Gln Tyr Trp Leu Arg 645 650 655Lys Pro Glu Leu Leu Leu Phe Ser Gln Met Tyr Ile Ser Asn Thr Lys 660 665 67057897DNAArabidopsis thaliana 57atggcatctg tttactccac cctaacctac tggctcgtcc accaccccta cattgccaac 60ttcacgtgga ccgaaggtga aacactaggc tccaccgttt tctttgtctt tgtcgtcgtc 120tccctttacc tctccgccac attcctcctc cgatacaccg tcgattcact ccccacactc 180ggtccccgca ttctcaaacc aatcacagcc gttcacagcc tcattctctt cctcctctcc 240ttaaccatgg ccgttggttg cactctctcc ctaatctctt cctcggaccc gaaggcgcgt 300ctcttcgacg ccgtttgttt ccccctcgac gtgaaaccta agggaccgct tttcttttgg 360gctcaagtct tttacctctc gaagatcctt gagttcgtag acacacttct catcatactc 420aacaaatcaa tccaacggct ctcgttcctc cacgtctacc accacgcaac ggttgtgatt 480ttgtgctacc tctggttacg aacacgtcaa tcgatgtttc ctgttgggct cgtgttgaac 540tcgacggtcc atgtgattat gtacgggtac tatttcctct gcgctatcgg atcgaggccc 600aagtggaaga agttggtgac gaattttcaa atggttcagt ttgctttcgg catggggtta 660ggagccgctt ggatgctccc agagcattat ttcgggtcgg gttgcgccgg gatttggaca 720gtttatttca atggtgtgtt tactgcttct ctattggctc tcttctacaa cttccactcc 780aagaactatg agaagactac aacgtcgcct ttgtataaga tcgaatcctt tatatttatt 840cacggagaga ggtgggcaaa taaagcgatt acattatttt ccaagaaaaa cgattaa 89758298PRTArabidopsis thaliana 58Met Ala Ser Val Tyr Ser Thr Leu Thr Tyr Trp Leu Val His His Pro1 5 10 15Tyr Ile Ala Asn Phe Thr Trp Thr Glu Gly Glu Thr Leu Gly Ser Thr 20 25 30Val Phe Phe Val Phe Val Val Val Ser Leu Tyr Leu Ser Ala Thr Phe 35 40 45Leu Leu Arg Tyr Thr Val Asp Ser Leu Pro Thr Leu Gly Pro Arg Ile 50 55 60Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Phe Leu Leu Ser65 70 75 80Leu Thr Met Ala Val Gly Cys Thr Leu Ser Leu Ile Ser Ser Ser Asp 85 90 95Pro Lys Ala Arg Leu Phe Asp Ala Val Cys Phe Pro Leu Asp Val Lys 100 105 110Pro Lys Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr Leu Ser Lys 115 120 125Ile Leu Glu Phe Val Asp Thr Leu Leu Ile Ile Leu Asn Lys Ser Ile 130 135 140Gln Arg Leu Ser Phe Leu His Val Tyr His His Ala Thr Val Val Ile145 150 155 160Leu Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe Pro Val Gly 165 170 175Leu Val Leu Asn Ser Thr Val His Val Ile Met Tyr Gly Tyr Tyr Phe 180 185 190Leu Cys Ala Ile Gly Ser Arg Pro Lys Trp Lys Lys Leu Val Thr Asn 195 200 205Phe Gln Met Val Gln Phe Ala Phe Gly Met Gly Leu Gly Ala Ala Trp 210 215 220Met Leu Pro Glu His Tyr Phe Gly Ser Gly Cys Ala Gly Ile Trp Thr225 230 235 240Val Tyr Phe Asn Gly Val Phe Thr Ala Ser Leu Leu Ala Leu Phe Tyr 245 250 255Asn Phe His Ser Lys Asn Tyr Glu Lys Thr Thr Thr Ser Pro Leu Tyr 260 265 270Lys Ile Glu Ser Phe Ile Phe Ile His Gly Glu Arg Trp Ala Asn Lys 275 280 285Ala Ile Thr Leu Phe Ser Lys Lys Asn Asp 290 29559837DNAArabidopsis thaliana 59atggcatcaa tttactcctc tttaacctac tggctcgtta accaccccta catctccaat 60tttacttgga tcgaaggtga aaccctaggc tccaccgtct ttttcgtatc cgtcgtagtc 120tccgtttacc tctccgccac gttcctcctc cgatccgcca tcgattcact cccatcactc 180agtccacgta tcctcaaacc gatcacagcc gtccacagcc taatcctctg tctcctctcc 240ttagtcatgg ccgtcggttg cactctctca ataacctcat ctcacgcgtc ttcagatccg 300atggcgcgtt tccttcacgc gatttgcttt cccgtcgacg ttaaacctaa cggaccgctt 360ttcttctggg ctcaagtctt ctacctctcg aagatcctcg agttcggaga cacgatcctc 420atcatactcg gcaaatcaat ccaacggcta tccttcctcc acgtgtacca ccacgcgacg 480gttgtggtca tgtgttatct ctggctccga actcgccaat cgatgtttcc gattgcgctc 540gtgacgaatt cgacggtaca cgtcatcatg tacggttact acttcctctg cgccgttgga 600tcgaggccca agtggaagag attggtgacg gattgtcaga ttgttcagtt tgttttcagt 660ttcgggttat ccggttggat gctccgagag cacttattcg ggtcgggttg caccgggatt 720tggggatggt gtttcaacgc tgcatttaat gcttctcttt tggctctctt ttccaacttc 780cattcaaaga attatgtcaa gaagccaacg agagaggatg gcaaaaaaag cgattag 83760278PRTArabidopsis thaliana 60Met Ala Ser Ile Tyr Ser Ser Leu Thr Tyr Trp Leu Val Asn His Pro1 5 10 15Tyr Ile Ser Asn Phe Thr Trp Ile Glu Gly Glu Thr Leu Gly Ser Thr 20 25 30Val Phe Phe Val Ser Val Val Val Ser Val Tyr Leu Ser Ala Thr Phe 35 40 45Leu Leu Arg Ser Ala Ile Asp Ser Leu Pro Ser Leu Ser Pro Arg Ile 50 55 60Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Cys Leu Leu Ser65 70 75 80Leu Val Met Ala Val Gly Cys Thr Leu Ser Ile Thr Ser Ser His Ala 85 90 95Ser Ser
Asp Pro Met Ala Arg Phe Leu His Ala Ile Cys Phe Pro Val 100 105 110Asp Val Lys Pro Asn Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr 115 120 125Leu Ser Lys Ile Leu Glu Phe Gly Asp Thr Ile Leu Ile Ile Leu Gly 130 135 140Lys Ser Ile Gln Arg Leu Ser Phe Leu His Val Tyr His His Ala Thr145 150 155 160Val Val Val Met Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe 165 170 175Pro Ile Ala Leu Val Thr Asn Ser Thr Val His Val Ile Met Tyr Gly 180 185 190Tyr Tyr Phe Leu Cys Ala Val Gly Ser Arg Pro Lys Trp Lys Arg Leu 195 200 205Val Thr Asp Cys Gln Ile Val Gln Phe Val Phe Ser Phe Gly Leu Ser 210 215 220Gly Trp Met Leu Arg Glu His Leu Phe Gly Ser Gly Cys Thr Gly Ile225 230 235 240Trp Gly Trp Cys Phe Asn Ala Ala Phe Asn Ala Ser Leu Leu Ala Leu 245 250 255Phe Ser Asn Phe His Ser Lys Asn Tyr Val Lys Lys Pro Thr Arg Glu 260 265 270Asp Gly Lys Lys Ser Asp 2756130DNAArtificial SequencePrimer 61gcggccgcca tgaagatata ctctagaacg 306227DNAArtificial SequencePrimer 62gatatcttag gctgcctctg caaaccc 276330DNAArtificial SequencePrimer 63gcggccgcca tgggagtata ctcgagagcg 306427DNAArtificial SequencePrimer 64cccgggtcac acggcttctg cgaagcc 276530DNAArtificial SequencePrimer 65gcggccgcca tgggaactag gttccaatca 306627DNAArtificial SequencePrimer 66aggcctttaa gcagctttgg cgaatcc 276731DNAArtificial SequencePrimer 67gagcggccgc catggagaaa ggtttgacga t 316831DNAArtificial SequencePrimer 68gagcggccgc cttaaggatg caagggctcc t 316935DNAArtificial SequencePrimer 69gagcggccgc catgcattgg catggtgtag agcag 357031DNAArtificial SequencePrimer 70gagcggccgc cttattcata gcaaggcggc a 317132DNAArtificial SequencePrimer 71gagcggccgc catgtctgct tctgattcct ct 327231DNAArtificial SequencePrimer 72gagcggccgc cttagtcgcg gaactcgtcc a 317334DNAArtificial SequencePrimer 73gagcggccgc catggcgttc cctaaggtat actt 347431DNAArtificial SequencePrimer 74gagcggccgc cctaagagag ctgaccacaa t 317531DNAArtificial SequencePrimer 75gagcggccgc catgggtagt gcaaaatcag c 317631DNAArtificial SequencePrimer 76gagcggccgc cttaggcgat ggagctttta t 317731DNAArtificial SequencePrimer 77gagcggccgc catgactaat cccatgatca t 317831DNAArtificial SequencePrimer 78gagcggccgc cctagagacg gtggatcaac g 317931DNAArtificial SequencePrimer 79gagcggccgc catggtttct tcttctttaa c 318031DNAArtificial SequencePrimer 80gagcggccgc cttaataatt ggtagcttta t 318131DNAArtificial SequencePrimer 81gagcggccgc catggccgga gttttcaaaa c 318231DNAArtificial SequencePrimer 82gagcggccgc ctcaaaagag agcaacaaca g 318331DNAArtificial SequencePrimer 83gagcggccgc catggcgtca aagcaactga g 318431DNAArtificial SequencePrimer 84gagcggccgc ctcacttgtt ggtgaacttt g 318531DNAArtificial SequencePrimer 85gcggccgcca tggcaacggg ggctgagaac c 318627DNAArtificial SequencePrimer 86aggcctttac cggcgaccac cagcagg 278730DNAArtificial SequencePrimer 87gcggccgcca tggcaaccat ggctaggtcg 308828DNAArtificial SequencePrimer 88aggccttcag ttgtcgtgca atgctttg 288930DNAArtificial SequencePrimer 89gcggccgcca tggcgcaacc cctcgtgaag 309027DNAArtificial SequencePrimer 90aggcctttag ccgctggcaa caatctc 279131DNAArtificial SequencePrimer 91gcggccgcca tgccgattag ccggagagtt c 319226DNAArtificial SequencePrimer 92gatatctcat atggaatcat aaaccg 269331DNAArtificial SequencePrimer 93gcggccgcca tgcccattaa tcagaaaatt c 319425DNAArtificial SequencePrimer 94aggccttcat ttgcgatcaa gaacc 259537DNAArtificial SequencePrimer 95ctgcaggcgg ccgccatgga gtgtagttca gtgagtg 379628DNAArtificial SequencePrimer 96aggcctctag tattggacta acgataac 289730DNAArtificial SequencePrimer 97gcggccgcat ggttgaaacc ttgtttgaag 309826DNAArtificial SequencePrimer 98aggcctctag gccttatcca ccttcc 269930DNAArtificial SequencePrimer 99gcggccgcat gggcaaaaaa gacatgccta 3010028DNAArtificial SequencePrimer 100aggccttcat attgtcgtgt aacgaggg 2810130DNAArtificial SequencePrimer 101gcggccgcat gatattatcg tttcgtggac 3010228DNAArtificial SequencePrimer 102aggcctctat acagatttgc catcgctc 2810329DNAArtificial SequencePrimer 103gcggccgcat gaaccggatg atcgaagcg 2910427DNAArtificial SequencePrimer 104aggccttcat atttggtgca cctcggc 2710528DNAArtificial SequencePrimer 105gcggccgcat gcagaccgtt tctcggag 2810626DNAArtificial SequencePrimer 106aggccttcaa ggataagact ctggag 2610729DNAArtificial SequencePrimer 107gcggccgcat ggcttcggtt actttctct 2910826DNAArtificial SequencePrimer 108aggccttcac ttccagttgt tggcaa 2610928DNAArtificial SequencePrimer 109gcggccgcat ggcaaaagaa aatggatt 2811026DNAArtificial SequencePrimer 110aggcctttag atagagaggt cagcga 2611128DNAArtificial SequencePrimer 111gcggccgcat ggcggcgaaa attcccgg 2811226DNAArtificial SequencePrimer 112aggccttcaa gacatgaaca gagcct 2611329DNAArtificial SequencePrimer 113gcggccgcat gggttacata ggagctcat 2911426DNAArtificial SequencePrimer 114aggccttcaa gcttctttac gcgtga 2611530DNAArtificial SequencePrimer 115gcggccgcat gtctccttct cactccatca 3011626DNAArtificial SequencePrimer 116aggccttcat ttggtgtttg aaatat 2611732DNAArtificial SequencePrimer 117gcggccgcat ggcatctgtt tactccaccc ta 3211826DNAArtificial SequencePrimer 118aggcctttaa tcgtttttct tggaaa 2611931DNAArtificial SequencePrimer 119gcggccgcat ggcatcaatt tactcctctt t 3112026DNAArtificial SequencePrimer 120aggcctctaa tcgctttttt tgccat 261211200DNAHelianthus annuus 121atgcagaccg tttctcggag attagctcgt gaaaatttga gctctcgcac atcgatttac 60tctctcaaat cgctttatcc tgtttccgat cgctgttacg gtgagtatga tcggcgttat 120gcctctacgc ttaccaccaa aggtattgga catctggtcc gcaagggtac tggtggaaga 180tcgtctgtta gtgggatagt tgctacagta ttcggagcta ctggtttcct tgggcgttac 240ttggtgcaac agcttgctaa aacgggttca caagtgctag taccatttag aggttccgaa 300gattcgcccc gtcatctcaa actgatgggc gatttggggc agattgttcc catgaaatat 360aatcctagag atgaaaactc aattaaggca gtcatggcca aggcaaatgt tgtgattaat 420ctcataggaa gggaatatga aaccagaaat tatagttttg aggaagtgaa ccatcatatg 480gctgaacaac ttgcaaagat ttccaaagaa catggtggaa tcatgagatt tatacaactg 540tcgtgtttag gtgcatctaa atcatctcca tctaggatgc ttcaagccaa ggctgctgca 600gaagaatcca tcttacgtga attgcctgag gccacaatac tgcgacctgc agtgatggtt 660ggtacagaag atcggatctt gaacccatgg gctcagttcg ctaaaaaata taactttctt 720ccaatgatcg ggggtggttc tactaagatt cagcctgtgt atgttgctga tgtcgcctct 780gcagttgttg cggcattaag tgatgacggt agtagcatgg gaaaagtgta tgaacttggt 840gggcctgatg tttatacact gcatcaattg gctgaactta tgtatgaaac gattcgagaa 900tggcctcatt atgttaacgt tcctttccct attgctaagg cgatctcaac acctcgagaa 960gtatttctta ataaagttcc cttcccgtta ccctcaccaa tcatcttcaa tttggatgtg 1020attaatgctc tttcttcaga tactctcgtc tcaaaagatg ctctgacatt caatgatctt 1080gagcttgtgc cacataaggt gaagggatat cctattgagt accttatcca gtatcgcaag 1140ggtggaccca attacggctc tacagtcagt gaaagagtga ctccagagtc ttatccttga 1200122399PRTHelianthus annuus 122Met Gln Thr Val Ser Arg Arg Leu Ala Arg Glu Asn Leu Ser Ser Arg1 5 10 15Thr Ser Ile Tyr Ser Leu Lys Ser Leu Tyr Pro Val Ser Asp Arg Cys 20 25 30Tyr Gly Glu Tyr Asp Arg Arg Tyr Ala Ser Thr Leu Thr Thr Lys Gly 35 40 45Ile Gly His Leu Val Arg Lys Gly Thr Gly Gly Arg Ser Ser Val Ser 50 55 60Gly Ile Val Ala Thr Val Phe Gly Ala Thr Gly Phe Leu Gly Arg Tyr65 70 75 80Leu Val Gln Gln Leu Ala Lys Thr Gly Ser Gln Val Leu Val Pro Phe 85 90 95Arg Gly Ser Glu Asp Ser Pro Arg His Leu Lys Leu Met Gly Asp Leu 100 105 110Gly Gln Ile Val Pro Met Lys Tyr Asn Pro Arg Asp Glu Asn Ser Ile 115 120 125Lys Ala Val Met Ala Lys Ala Asn Val Val Ile Asn Leu Ile Gly Arg 130 135 140Glu Tyr Glu Thr Arg Asn Tyr Ser Phe Glu Glu Val Asn His His Met145 150 155 160Ala Glu Gln Leu Ala Lys Ile Ser Lys Glu His Gly Gly Ile Met Arg 165 170 175Phe Ile Gln Leu Ser Cys Leu Gly Ala Ser Lys Ser Ser Pro Ser Arg 180 185 190Met Leu Gln Ala Lys Ala Ala Ala Glu Glu Ser Ile Leu Arg Glu Leu 195 200 205Pro Glu Ala Thr Ile Leu Arg Pro Ala Val Met Val Gly Thr Glu Asp 210 215 220Arg Ile Leu Asn Pro Trp Ala Gln Phe Ala Lys Lys Tyr Asn Phe Leu225 230 235 240Pro Met Ile Gly Gly Gly Ser Thr Lys Ile Gln Pro Val Tyr Val Ala 245 250 255Asp Val Ala Ser Ala Val Val Ala Ala Leu Ser Asp Asp Gly Ser Ser 260 265 270Met Gly Lys Val Tyr Glu Leu Gly Gly Pro Asp Val Tyr Thr Leu His 275 280 285Gln Leu Ala Glu Leu Met Tyr Glu Thr Ile Arg Glu Trp Pro His Tyr 290 295 300Val Asn Val Pro Phe Pro Ile Ala Lys Ala Ile Ser Thr Pro Arg Glu305 310 315 320Val Phe Leu Asn Lys Val Pro Phe Pro Leu Pro Ser Pro Ile Ile Phe 325 330 335Asn Leu Asp Val Ile Asn Ala Leu Ser Ser Asp Thr Leu Val Ser Lys 340 345 350Asp Ala Leu Thr Phe Asn Asp Leu Glu Leu Val Pro His Lys Val Lys 355 360 365Gly Tyr Pro Ile Glu Tyr Leu Ile Gln Tyr Arg Lys Gly Gly Pro Asn 370 375 380Tyr Gly Ser Thr Val Ser Glu Arg Val Thr Pro Glu Ser Tyr Pro385 390 3951231653DNAArabidopsis thaliana 123atggagtgta gttcagtgag tgtactagga atattactgg tatttcctct ccttcataac 60cttgtcacca tctccgggca gaatcttccg gcggtgggtt tgttcacttt cggagattcc 120aacttcgacg ctggaaataa aaagttcctc acaagtgctc cacttcctca aaacttttgg 180ccttacggta aatctcgaga tgaccctaag ggcaagtttt ctgatggcaa aattgtcccg 240gactttattg caaaattcat ggggatacca cacgatttac cgccggcgct aaaacccggc 300accgatgtgt cacgaggagc cagcttcgcc gtcgggtccg cttccattct tggatctcca 360aaagattctt tggctctgaa tcaacaagtg aggaaattca atcagatgat atcaaattgg 420aaagtggatt acattcagaa atcagtgttt atgattagca ttggtatgga agattactac 480aactttacca aaaacaatcc taatgctgaa gtttctgctc aacaagcttt cgttacttct 540gtcactaacc ggtttaagag tgatatcaac ttgttgtatt catctggagc tagtaaattc 600gtcgtacact tgctagcgcc attaggttgt ttaccgatcg caagacaaga atttaaaacc 660ggtaacaatt gttacgagaa actcgatgat ttggccaaac aacacaacgc taaaattgga 720ccgattttga acgaaatggc ggaaactaaa ccggatttcc aattcaccgt tttcgatttc 780tacaacgtta ttcttcgcag gacacaaaga aacatgaact accggttttc cgtgacgaat 840atatcgtgtt gcggtgttgg gacgcattat gcatatggtt gtggtttacc taacgtgcac 900tcgaagttat gcgaatatca aagatcctac ctttacttcg acgcacgtca taacacagag 960aaagcacaag aagcgtttgc tcatcttatc tttggagctg acccaaatgt tatccaacct 1020atgaatgttc gtgagctcat ggtgtatcct gttaatgagc ctatgcgtga gttttgggag 1080gatccaatgg atgagaagtt atcgttagtc caatactaga ggagcttgtt gagcagaaac 1140ttcagcatta ggattgtttt tggtaaagtt gtagtaatct tccataccaa tgctaatcat 1200aaacactgat ttctgaatgt aatccacttt ccaatttgat atcatctgat tgaatttcct 1260cacttgttga ttcagagcca aagaatcttt tggagatcca agaatggaag cggacccgac 1320ggcgaagctg gctcctcgtg acacatcggt gccgggtttt agcgccggcg gtaaatcgtg 1380tggtatcccc atgaattttg caataaagtc cgggacaatt ttgccatcag aaaacttgcc 1440cttagggtca tctcgagatt taccgtaagg ccaaaagttt tgaggaagtg gagcacttgt 1500gaggaacttt ttatttccag cgtcgaagtt ggaatctccg aaagtgaaca aacccaccgc 1560cggaagattc tgcccggaga tggtgacaag gttatgaagg agaggaaata ccagtaatat 1620tcctagtaca ctcactgaac tacactccat ggc 1653
Patent applications by JÖrg Bauer, Ludwigshafen DE
Patent applications by JÖrg Bauer, Ludwigshafen DE
Patent applications by Oliver Oswald, Ludwigshafen DE
Patent applications by Thorsten Zank, Mannheim DE
Patent applications by BASF Plant Science GmbH
Patent applications in class The polynucleotide alters fat, fatty oil, ester-type wax, or fatty acid production in the plant
Patent applications in all subclasses The polynucleotide alters fat, fatty oil, ester-type wax, or fatty acid production in the plant