Plastidic Phosphoglucomutase Genes

Abstract

An isolated nucleic acid fragment encoding a plastidic phosphoglucomutase protein is disclosed. Also disclosed is the construction of a chimeric gene encoding all or a substantial portion of the plastidic phosphoglucomutase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the plastidic phosphoglucomutase in a transformed host cell.

Description

[0001] This application is a Continuation of U.S. patent application No.-in-Part and claims the benefit of U.S. patent application Ser. No. 09/906,209, filed Jul. 16, 2001, now pending, and U.S. Provisional Application No. 60/218,712, filed Jul. 17, 2000, now expired, the entire contents of both are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to isolated nucleic acid fragments encoding plastidic phosphoglucomutase proteins in plants and seeds and the use of such fragments to modulate expression of a gene encoding plastidic phosphoglucomutase activity.

BACKGROUND OF THE INVENTION

[0003] Starch synthesis occurs in the chloroplast while soluble carbohydrate (i.e., sucrose) synthesis occurs in the cytosol. These biosynthetic pathways are competing processes because excess triose phosphate can be used for either starch synthesis in the chloroplast or sucrose synthesis in the cytosol. These pathways have many common steps, however, the enzymes that catalyze similar steps are unique to each compartment. These enzymes are isozymes; different forms of the enzymes that catalyze the same reaction. For example, the plastidic and cytosolic forms of phosphoglucomutase both catalyze the conversion of glucose-6-phosphate to glucose 1-phosphate in different subcellular locations.

[0004] At maturity, about 40% of soybean seed dry weight is protein and 20% extractable oil. These constitute the economically valuable products of the soybean crop. Of the remaining 40% of seed weight, about 10% is soluble carbohydrate. The soluble carbohydrate portion contributes little to the economic value of soybean seeds and the main component of the soluble carbohydrate fraction, raffinosaccharides, are deleterious both to processing and to the food value of soybean meal in monogastric animals (Coon et al., (1988) Proceedings Soybean Utilization Alternatives, Univ. of Minnesota, pp. 203-211).

[0005] It may be possible to modulate the size of the starch and soluble carbohydrate pools in plant cells by altering the catalytic activity of specific enzymes in the starch and soluble carbohydrate biosynthetic pathways, such as phosphoglucomutase or one or both of the large and small subunits of ADP-glucose pyrophosphorylase (Taiz L., et al. Plant Physiology; The Benjamin/Cummings Publishing Company: New York, 1991). For example, during soybean seed maturation a large portion of the glucose which is converted to soluble carbohydrates (sucrose, raffinose and stachyose) during soybean seed maturation comes from the break down of a starch pool which was produced slowly during the primary growth phase. Elimination of this transient starch pool may be a strategy for diverting carbon away from the soluble carbohydrate components of dry soybean seeds (sucrose, raffinose and stachyose) and into the more economically desirable components such as oil and protein. This strategy may also be applicable to other plants such as corn, rice and wheat. Elimination of ADP-glucose pyrophosphorylase expression in developing maize embryos may decrease the production of transient starch in that tissue and lead to a concomitant increase in the oil content of the embryo [Singletary, G et al. (2001) U.S. Pat. No. 6,232,529].

[0006] There is a great deal of interest in identifying the genes that encode proteins involved in starch and soluble carbohydrate biosynthesis in plants. The genes that code for these enzymes may be used to study the interactions among individuals of the pathways and develop methods to alter starch and soluble carbohydrate biosynthesis. Accordingly, the availability of nucleic acid sequences encoding all or a substantial portion of a plastidic or cytosolic phosphoglucomutase (PGM) enzyme would facilitate studies to better understand starch and soluble carbohydrate biosynthesis in plants and provide genetic tools to enhance or otherwise alter starch and soluble carbohydrate biosynthesis.

[0007] Previous reports on a plastidic PGM mutant (pgm-1) from the oilseed plant Arabidopsis (Caspar et al. (1985) Plant Physiol. 79:11-17; Periappuram et al., (2000) Plant Physiol. 122:1193-1199) indicated that pgm-1 mutant plants showed a decrease in seed lipid content and an increase in leaf soluble carbohydrates. High levels of soluble carbohydrates were also observed in starchless Nicotiana sylvestris plants deficient in the plastidic PGM activity (Huber and Hanson, (1992) Plant Physiol. 99:1449-1454). Yet another effect of reduced starch content on carbon partitioning was observed in pea (Pisum sativum). Seeds from wild type pea typically contain 60% of the seed dry weight as starch. The rug3 locus of Pisum sativum encodes the pea plastidic phosphoglucomutase. Pea seeds, of the rug3rug3 genotype, substantially lacking plastidic phosphoglucomutase activity, have a wrinkled phenotype, higher levels of sucrose and an increased lipid content at maturity (EP 1001029A1; Casey et al., (1998) J. Plant Physiol. 152: 636-640).

SUMMARY OF THE INVENTION

[0008] In a first embodiment, the present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 560 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:8 have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 560 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, or (c) the complement of the first or second nucleotide sequence, wherein the complement and the first or second nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:8, and the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:7, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:9. The first and second polypeptides preferably have phosphoglucomutase activity.

[0009] In a second embodiment, the present invention relates to a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence.

[0010] In a third embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.

[0011] In a fourth embodiment, the present invention relates to an isolated polynucleotide fragment comprising a nucleotide sequence comprised by any of the polynucleotides of the present invention, wherein the nucleotide sequence contains at least 30, 40, 60, 100, 200, 300, 400, 500 or 541 nucleotides.

[0012] In a fifth embodiment, the present invention relates to a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0013] In a sixth embodiment, the invention concerns a method for transforming a cell, comprising transforming a cell with a polynucleotide of the present invention.

[0014] In a seventh embodiment, the present invention relates to a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a transgenic plant from the transformed plant cell.

[0015] In an eighth embodiment, the invention concerns a cell, plant, or seed comprising a recombinant DNA construct of the present invention.

[0016] In a ninth embodiment, the present invention concerns an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 560 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:8 have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, and (b) a second amino acid sequence comprising at least 560 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:8, and the second amino acid sequence preferably comprises the amino acid sequence SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10. The polypeptide preferably has phosphoglucomutase activity.

[0017] In a tenth embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the recombinant DNA constructs of the present invention.

[0018] In an eleventh embodiment, the invention relates to a method of selecting an isolated polynucleotide that alters, i.e., increases or decreases, the level of expression of a phosphoglucomutase gene, protein or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; (b) introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; (c) measuring the level of the phosphoglucomutase RNA, protein or enzyme activity in the host cell containing the isolated polynucleotide or recombinant DNA construct; (d) comparing the level of the phosphoglucomutase RNA, protein or enzyme activity in the host cell containing the isolated polynucleotide or recombinant DNA construct with the level of the phosphoglucomutase RNA, protein or enzyme activity in a host cell that does not contain the isolated polynucleotide or recombinant DNA construct, and selecting the isolated polynucleotide or recombinant DNA construct that alters, i.e., increases or decreases, the level of expression of the phosphoglucomutase gene, protein or enzyme activity in the plant cell.

[0019] In a twelfth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a phosphoglucomutase protein, preferably a plant phosphoglucomutase protein, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, more preferably at least 60 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, and 9, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a phosphoglucomutase protein amino acid sequence.

[0020] In a thirteenth embodiment, the invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a phosphoglucomutase protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

[0021] In a fourteenth embodiment, the invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the phosphoglucomutase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

[0022] In a fifteenth embodiment, this invention concerns a method for suppressing the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity in a transgenic plant, wherein the method comprises: [0023] (a) transforming a plant cell with a fragment of the isolated polynucleotide of the invention; [0024] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0025] (c) selecting a transgenic plant wherein the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity has been suppressed.

[0026] Preferably, the gene encodes a plastidic polypeptide having phosphoglucomutase activity, and the plant is a soybean plant.

[0027] In a sixteenth embodiment, the invention concerns a method for producing transgenic seed, the method comprising: [0028] (a) transforming a plant cell with the recombinant DNA construct of [0029] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0030] (ii) the complement of (i); [0031] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0032] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0033] (c) selecting a transgenic plant that produces a transgenic seed having an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% as compared to seed obtained from a non-transgenic plant.

[0034] Preferably, the seed is a soybean seed.

[0035] In a seventeenth embodiment, the invention concerns a method method for producing transgenic seed, the method comprising: [0036] (a) transforming a plant cell with a recombinant DNA construct comprising [0037] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0038] (ii) the complement of (i); [0039] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0040] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0041] (c) selecting a transgenic plant that produces a transgenic seed having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to seed obtained from a non-transgenic plant.

[0042] Preferably, the transgenic seed differs from an untransformed seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%. Preferably, the seed is a soybean seed.

[0043] In an eighteenth embodiment, the invention concerns a method for producing defatted meal from transgenic seed, comprising: [0044] (a) transforming a plant cell with a recombinant DNA construct comprising [0045] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0046] (ii) the complement of (i); [0047] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0048] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0049] (c) selecting a transgenic plant that produces a transgenic seed wherein said seed is processed into defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% as compared to defatted meal obtained from seed of a non-transgenic plant.

[0050] Preferably, the defatted meal of the transgenic seed differs from the defatted meal of an untransformed seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the seed is a soybean seed.

[0051] In a nineteenth embodiment, the invention concerns a transgenic seed that differs from an non-transgenic seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%, and a decrease in the sucrose content of at least 25%. Preferably, the seed is a soybean seed.

[0052] In a twentieth embodiment, the invention concerns a transgenic seed that differs from non-transgenic seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the transgenic seed differs from an untransformed seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%. Preferably, the seed is a soybean seed. In a twenty-first embodiment, the invention concerns a transgenic seed comprising a recombinant construct comprising (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or

[0053] (ii) the complement of (i);

[0054] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant;

[0055] further wherein said transgenic seed is processed to make defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% when compared to defatted meal obtained from a non-transgenic seed.

[0056] Preferably, the defatted meal of the transgenic seed differs from the defatted meal of an untransformed seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the seed is a soybean seed.

[0057] In a twenty-second embodiment, the invention concerns a recombinant DNA construct comprising: (a) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or (b) the complement of (a); wherein (a) or (b) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING

[0058] The invention can be more fully understood from the following detailed description and the accompanying Drawing and Sequence Listing which form a part of this application.

[0059] FIGS. 1A-1D show an alignment of the amino acid sequences of plastidic phosphoglucomutase encoded by the nucleotide sequences derived from the following: cattail clone etr1c.pk005.f8 (SEQ ID NO:2); corn contig (SEQ ID NO:4) composed of p0075.cslaf22f (EST), p0075.cslaf22rb (EST), and p0128.cpicz81r (EST); soybean contig (SEQ ID NO:8) composed of clone sdp3c.pk003.e22 and PCR fragments; rice clone rdi1c.pk001.a22 (SEQ ID NO:10); plastidic phosphoglucomutase from Brassica napus (NCBI General Identifier No. 6272125; SEQ ID NO:11); plastidic phosphoglucomutase from Pisum sativum (NCBI General Identifier No. 6272283; SEQ ID NO:12); and plastidic phosphoglucomutase from Pisum sativum described in European Patent Application EP 1001029-A (NCBI General Identifier No. 10190529; SEQ ID NO:13). For the consensus alignment, amino acids which are conserved among all sequences at a given position, and which are contained in at least two sequences, are indicated with an asterisk (*). Dashes are used by the program to maximize alignment of the sequences. Amino acid positions for a given SEQ ID NO are given to the left of the corresponding line of sequence. Amino acid positions for the consensus alignment are given below each section of sequence.

[0060] FIG. 2 shows the starch accumulation expressed as mg/g fresh weight (top) and mg/seed (bottom) in plastidic PGM-silenced seeds (ko) as compared to wild-type seeds (wt).

[0061] FIG. 3 shows the soluble carbohydrate concentrations of growth chamber (GC) or field grown (field) T2 seeds from plastidic PGM-silenced events (KO) as compared to their null segregants (WT).

[0062] FIG. 4 shows soluble carbohydrate concentrations of T3 seeds from plastidic PGM-silenced events as compared to wild-type. PGM-silenced seeds with a 92B91 genetic background were compared to a 92B91 null event, while PGM-silenced seeds with a Jack background were compared to a Jack null event.

[0063] FIG. 5 shows the soluble carbohydrate profile of defatted soybean meal from T2 seeds.

[0064] Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*", or sequences encoding the entire or functional protein derived from an FIS, a contig, an EST and PCR, or an FIS and PCR ("CGS"). Nucleotide SEQ ID NOs:1, 3, 5, and 7 correspond to nucleotide SEQ ID NOs:1, 3, 5, and 7, respectively, presented in U.S. Provisional Application No. 60/218,712, filed Jul. 17, 2000. Amino acid SEQ ID NOs:2, 4, 6, and 8 correspond to amino acid SEQ ID NOs:2, 4, 6, and 8, respectively, presented in U.S. Provisional Application No. 60/218,712, filed Jul. 17, 2000. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37C.F.R. .sctn.1.821-1.825.

TABLE-US-00001 TABLE 1 Plastidic Phosphoglucomutase Proteins SEQ ID NO: Protein Amino (Plant Source) Clone Designation Status Nucleotide Acid Plastidic etr1c.pk005.f8 (FIS) CGS 1 2 Phospho- glucomutase (Cattail) Plastidic Contig Composed of: CGS 3 4 Phospho- p0075.cslaf22f (EST); glucomutase p0075.cslaf22rb (Corn) (EST); p0128.cpicz81r (EST) Plastidic rth1c.pk009.k14.f EST 5 6 Phospho- (EST) glucomutase (Rice) Plastidic Contig Composed of: CGS 7 8 Phospho- sdp3c.pk003.e22 glucomutase (EST); PCR (Soybean) Fragments Plastidic rdi1c.pk001.a22 (FIS) CGS 9 10 Phospho- glucomutase (Rice)

[0065] SEQ ID NO:10 corresponds to a direct translation of the nucleotide sequence for the full insert of rice clone rdi1c.pk001.a22. The amino acid sequence in SEQ ID NO:10 includes a 46 amino acid open-reading frame directly in front of, and in frame with, the methionine start codon.

[0066] SEQ ID NO:11 corresponds to plastidic phosphoglucomutase from Brassica napus (NCBI General Identifier No. 6272125).

[0067] SEQ ID NO:12 corresponds to plastidic phosphoglucomutase from Pisum sativum (NCBI General Identifier No. 6272283).

[0068] SEQ ID NO:13 corresponds to and plastidic phosphoglucomutase from Pisum sativum described in European Patent Application EP 1001029-A (NCBI General Identifier No. 10190529).

[0069] SEQ ID NO:14 corresponds to a 574 nucleotide NotI fragment from plasmid pTC103; this fragment contains a 541 nucleotide region of soybean plastidic phosphoglucomutase, a 19 nucleotide artificial sequence at the 5' end and a 14 nucleotide artificial sequence at the 3' end.

[0070] SEQ ID NO:15 corresponds to the 541 nucleotide region of soybean plastidic phosphoglucomutase contained in SEQ ID NO:14.

[0071] SEQ ID NO:16 corresponds to the full-insert sequence (FIS) of corn clone p0075.cslaf22rb.

[0072] SEQ ID NO:17 corresponds to the nucleotide sequence of plasmid pKS133.

[0073] SEQ ID NO:18 corresponds to a synthetic DNA linker.

[0074] SEQ ID NO:19 corresponds to synthetic complementary region of pKS106 and pKS124.

[0075] SEQ ID NO:20 corresponds to a synthetic complementary region of pKS133.

[0076] SEQ ID NO:21 corresponds to a synthetic PCR primer.

[0077] SEQ ID NO:22 corresponds to a synthetic PCR primer.

[0078] SEQ ID NO:23 corresponds to a nucleotide sequence of a contig made from the full-insert sequences of the cDNA inserts of soybean clones ses4d.pk0018.d10 and sdp2c.pk008.m2. The first 107 nucleotides of the contig were obtained from the sequence of clone ses4d.pk0018.d10.

[0079] SEQ ID NO:24 corresponds to the amino acid sequence of a large subunit polypeptide of soybean ADP-glucose pyrophosphorylase, and is encoded by nucleotides 42-1637 of SEQ ID NO:23.

[0080] SEQ ID NO:25 corresponds to the amino acid sequence of the large subunit of ADP-glucose pyrophosphorylase from chickpea, Cicer arietinum (NCBI General Identifier No. 13487785).

[0081] SEQ ID NO:26 corresponds to the amino acid sequence of SEQ ID NO:248406 from U.S. Patent Application US2004031072.

[0082] SEQ ID NO:27 corresponds to a nucleotide sequence obtained from the full-length sequence of the cDNA insert of soybean clone ssm.pk0072.e7:fis.

[0083] SEQ ID NO:28 corresponds to the amino acid sequence of a first small subunit polypeptide, SS1, of the soybean ADP-glucose pyrophosphorylase, and is encoded by nucleotides 80-1627 of SEQ ID NO:27.

[0084] SEQ ID NO:29 corresponds to a nucleotide sequence of a contig made from the EST sequence of soybean clone ssl.pk0021.h3 and the full-insert sequence of soybean clone sgs4c.pk005.b10. The first 58 nucleotides of the contig were obtained from the sequence of clone ssl.pk0021.h3.

[0085] SEQ ID NO:30 corresponds to the amino acid sequence of a second small subunit polypeptide, SS2, of the soybean ADP-glucose pyrophosphorylase, and is encoded by nucleotides 47-1594 of SEQ ID NO:29.

[0086] SEQ ID NO:31 corresponds to the amino acid sequence of the small subunit, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (NCBI General Identifier No. 29421116).

[0087] SEQ ID NO:32 corresponds to the amino acid sequence of SEQ ID NO:251944 from U.S. Patent Application US2004031072.

[0088] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn.1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0089] All patents, patent applications, and publications cited throughout the application are hereby incorporated by reference in their entirety.

[0090] In the context of this disclosure, a number of terms shall be utilized.

[0091] The term "raffinose family oligosaccharides" (RFOs) indicates a group of D-galactose containing oligosaccharides that are synthesized by a set of galactosyltransferases. Raffinose family oligosaccharides are characterized by having the general formula: O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n-.alpha.-D-glucopyranosyl-(- 1.fwdarw.2)-.beta.-D-fructofuranoside, where oligosaccharides with n=1 through n=4 are known respectively as raffinose, stachyose, verbascose, and ajugose. Examples of raffinose family oligosaccharides include, but are not limited to, raffinose, stachyose, verbascose and ajugose.

[0092] The term "plant" includes reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same. Plant cell, as used herein includes, without limitation, cells obtained from or found in the following: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.

[0093] Examples of monocots include, but are not limited to, corn, wheat, rice, sorghum, millet, barley, palm, lily, Alstroemeria, rye, and oat.

[0094] Examples of dicots include, but are not limited to, soybean, rape, sunflower, canola, grape, guayule, columbine, cotton, tobacco, peas, beans, flax, safflower, and alfalfa.

[0095] Plant tissue includes differentiated and undifferentiated tissues or plants, including but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue, and various forms of cells and culture such as single cells, protoplasm, embryos, and callus tissue. The plant tissue may in plant or in organ, tissue or cell culture.

[0096] The term "plant organ" refers to plant tissue or group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the following: 1. The entire complement of genetic material (genes and non-coding sequences) is present in each cell of an organism, or virus or organelle. 2. A complete set of chromosomes inherited as a (haploid) unit from one parent. The term "stably integrated" refers to the transfer of a nucleic acid fragment into the genome of a host organism or cell resulting in genetically stable inheritance.

[0097] The terms "polynucleotide", "polynucleotide sequence", "nucleic acid", nucleic acid sequence", and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.

[0098] The term "isolated" refers to materials, such as "isolated nucleic acid fragments" and/or "isolated polypeptides", which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

[0099] The term "isolated nucleic acid fragment" is used interchangeably with "isolated polynucleotide" and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.

[0100] The terms "subfragment that is functionally equivalent" and "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. For example, the fragment or subfragment can be used in the design of recombinant DNA constructs to produce the desired phenotype in a transformed plant. Recombinant DNA constructs can be designed for use in co-suppression or antisense by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the appropriate orientation relative to a plant promoter sequence.

[0101] "Cosuppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar native genes (U.S. Pat. No. 5,231,020). Cosuppression technology constitutes the subject matter of U.S. Pat. No. 5,231,020, which issued to Jorgensen et al. on Jul. 27, 1999. The phenomenon observed by Napoli et al. in petunia was referred to as "cosuppression" since expression of both the endogenous gene and the introduced transgene were suppressed (for reviews see Vaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).

[0102] Co-suppression constructs in plants previously have been designed by focusing on overexpression of a nucleic acid sequence having homology to an endogenous mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al. (1998) Plant J 16:651-659; and Gura (2000) Nature 404:804-808). The overall efficiency of this phenomenon is low, and the extent of the RNA reduction is widely variable. Recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication WO 99/53050 published on Oct. 21, 1999). This increases the frequency of co-suppression in the recovered transgenic plants. Another variation describes the use of plant viral sequences to direct the suppression, or "silencing", of proximal mRNA encoding sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998). Both of these co-suppressing phenomena have not been elucidated mechanistically, although recent genetic evidence has begun to unravel this complex situation (Elmayan et al. (1998) Plant Cell 10:1747-1757).

[0103] In addition to cosuppression, antisense technology has also been used to block the function of specific genes in cells. Antisense RNA is complementary to the normally expressed RNA, and presumably inhibits gene expression by interacting with the normal RNA strand. The mechanisms by which the expression of a specific gene are inhibited by either antisense or sense RNA are on their way to being understood. However, the frequencies of obtaining the desired phenotype in a transgenic plant may vary with the design of the construct, the gene, the strength and specificity of its promoter, the method of transformation and the complexity of transgene insertion events (Baulcombe, Curr. Biol. 12(3):R82-84 (2002); Tang et al., Genes Dev. 17(1):49-63 (2003); Yu et al., Plant Cell. Rep. 22(3):167-174 (2003)). Cosuppression and antisense inhibition are also referred to as "gene silencing", "post-transcriptional gene silencing" (PTGS), RNA interference or RNAi. See for example U.S. Pat. No. 6,506,559.

[0104] MicroRNAs (miRNA) are small regulatory RNSs that control gene expression. miRNAs bind to regions of target RNAs and inhibit their translation and, thus, interfere with production of the polypeptide encoded by the target RNA. miRNAs can be designed to be complementary to any region of the target sequence RNA including the 3' untranslated region, coding region, etc. miRNAs are processed from highly structured RNA precursors that are processed by the action of a ribonuclease III termed DICER. While the exact mechanism of action of miRNAs is unknown, it appears that they function to regulate expression of the target gene. See, e.g., U.S. Patent Publication No. 2004/0268441 A1 which was published on Dec. 30, 2004.

[0105] The term "expression", as used herein, refers to the production of a functional end-product, be it mRNA or translation of mRNA into a polypeptide. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020).

[0106] "Overexpression" refers to the production of a functional end-product in transgenic organisms that exceeds levels of production when compared to expression of that functional end-product in a normal, wild type or non-transformed organism.

[0107] "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. The preferred method of cell transformation of rice, corn and other monocots is using particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050), or an Agrobacterium-mediated method (Ishida Y. et al. (1996) Nature Biotech. 14:745-750). The term "transformation" as used herein refers to both stable transformation and transient transformation.

[0108] "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.

[0109] As stated herein, "suppression" refers to the reduction of the level of enzyme activity or protein functionality detectable in a transgenic plant when compared to the level of enzyme activity or protein functionality detectable in a plant with the native enzyme or protein. The level of enzyme activity in a plant with the native enzyme is referred to herein as "wild type" activity. The level of protein functionality in a plant with the native protein is referred to herein as "wild type" functionality. The term "suppression" includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. This reduction may be due to the decrease in translation of the native mRNA into an active enzyme or functional protein. It may also be due to the transcription of the native DNA into decreased amounts of mRNA and/or to rapid degradation of the native mRNA. The term "native enzyme" refers to an enzyme that is produced naturally in the desired cell.

[0110] "Gene silencing," as used herein, is a general term that refers to decreasing mRNA levels as compared to wild-type plants, does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression and stem-loop suppression.

[0111] The terms "homology", "homologous", "substantially similar" and "corresponding substantially" are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. For example, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes that result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences.

[0112] Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under moderately stringent conditions (for example, 1.times.SSC, 0.1% SDS, 60.degree. C.) with the sequences exemplified herein, or to any portion of the nucleotide sequences reported herein and which are functionally equivalent to the gene or the promoter of the invention. Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions involves a series of washes starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more preferred set of stringent conditions involves the use of higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2.times.SSC, 0.5% SDS was increased to 60.degree. C. Another preferred set of highly stringent conditions involves the use of two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

[0113] With respect to the degree of substantial similarity between the target (endogenous) mRNA and the RNA region in the construct having homology to the target mRNA, such sequences should be at least 25 nucleotides in length, preferably at least 50 nucleotides in length, more preferably at least 100 nucleotides in length, again more preferably at least 200 nucleotides in length, and most preferably at least 300 nucleotides in length; and should be at least 80% identical, preferably at least 85% identical, more preferably at least 90% identical, and most preferably at least 95% identical.

[0114] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least 70% identical, preferably at least 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least 95% identical to the amino acid sequences reported herein.

[0115] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying related polypeptide sequences. Useful examples of percent identities are 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%.

[0116] Sequence alignments and percent similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table on the same program.

[0117] Unless otherwise stated, "BLAST" sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0118] Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=.sup.-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0119] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0120] Thus, "Percentage of sequence identity" refers to the valued determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%. These identities can be determined using any of the programs described herein.

[0121] Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Megalign program of the LASARGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences are performed using the Clustal V method of alignment (Higgins, D. G. and Sharp, P. M. (1989) Comput. Appl. Biosci. 5:151-153; Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

[0122] It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other plant species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100%. Indeed, any integer amino acid identity from 50%-100% may be useful in describing the present invention. Also, of interest is any full or partial complement of this isolated nucleotide fragment.

[0123] The term "recombinant" means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.

[0124] As used herein, "contig" refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

[0125] "Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0126] The terms "synthetic nucleic acid" or "synthetic genes" refer to nucleic acid molecules assembled either in whole or in part from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. "Chemically synthesized", as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0127] "Gene" refers to a nucleic acid fragment that is capable of directing expression a specific protein or functional RNA.

[0128] "Native gene" refers to a gene as found in nature with its own regulatory sequences.

[0129] "Chimeric gene" or "recombinant DNA construct" are used interchangeably herein, and refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature, or to an isolated native gene optionally modified and reintroduced into a host cell.

[0130] A chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. In one embodiment, a regulatory region and a coding sequence region are assembled from two different sources. In another embodiment, a regulatory region and a coding sequence region are derived from the same source but arranged in a manner different than that found in nature. In another embodiment, the coding sequence region is assembled from at least two different sources. In another embodiment, the coding region is assembled from the same source but in a manner not found in nature.

[0131] The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism.

[0132] The term "foreign gene" refers to a gene not normally found in the host organism that is introduced into the host organism by gene transfer.

[0133] The term "transgene" refers to a gene that has been introduced into a host cell by a transformation procedure. Transgenes may become physically inserted into a genome of the host cell (e.g., through recombination) or may be maintained outside of a genome of the host cell (e.g., on an extrachromasomal array).

[0134] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.

[0135] The term "coding sequence" refers to a DNA fragment that codes for a polypeptide having a specific amino acid sequence, or a structural RNA. The boundaries of a protein coding sequence are generally determined by a ribosome binding site (prokaryotes) or by an ATG start codon (eukaryotes) located at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0136] "Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed. "Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.

[0137] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to a DNA that is complementary to and synthesized from an mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

[0138] The term "endogenous RNA" refers to any RNA which is encoded by any nucleic acid sequence present in the genome of the host prior to transformation with the recombinant construct of the present invention, whether naturally-occurring or non-naturally occurring, i.e., introduced by recombinant means, mutagenesis, etc.

[0139] The term "non-naturally occurring" means artificial, not consistent with what is normally found in nature.

[0140] "Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell.

[0141] "cDNA" refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.

[0142] "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro.

[0143] "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA, and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.

[0144] "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated, yet has an effect on cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts, and are meant to define the antisense RNA of the message.

[0145] The term "recombinant DNA construct" refers to a DNA construct assembled from nucleic acid fragments obtained from different sources. The types and origins of the nucleic acid fragments may be very diverse.

[0146] A "recombinant expression construct" contains a nucleic acid fragment operably linked to at least one regulatory element, that is capable of effecting expression of the nucleic acid fragment. The recombinant expression construct may also affect expression of a homologous sequence in a host cell.

[0147] In one embodiment the choice of recombinant expression construct is dependent upon the method that will be used to transform host cells. The skilled artisan is well aware of the genetic elements that must be present on the recombinant expression construct in order to successfully transform, select and propagate host cells. The skilled artisan will also recognize that different independent transformation events may be screened to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by, but is not limited to, Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0148] The term "operably linked" refers to the association of nucleic acid fragments on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA.

[0149] "Regulatory sequences" refer to nucleotides located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which may influence the transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences may include, and are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0150] "Promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoter sequences can also be located within the transcribed portions of genes, and/or downstream of the transcribed sequences. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of an isolated nucleic acid fragment in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause an isolated nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0151] Specific examples of promoters that may be useful in expressing the nucleic acid fragments of the invention include, but are not limited to, the oleosin promoter (PCT Publication WO99/65479, published Dec. 12, 1999), the maize 27 kD zein promoter (Ueda et al (1994) Mol. Cell. Biol. 14:4350-4359), the ubiquitin promoter (Christensen et al (1992) Plant Mol. Biol. 18:675-680), the SAM synthetase promoter (PCT Publication WO00/37662, published Jun. 29, 2000), the CaMV 35S (Odell et al (1985) Nature 313:810-812), and the promoter described in PCT Publication WO02/099063 published Dec. 12, 2002.

[0152] The "translation leader sequence" refers to a polynucleotide fragment located between the promoter of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D. (1995) Mol. Biotechnol. 3:225-236).

[0153] An "intron" is an intervening sequence in a gene that does not encode a portion of the protein sequence. Thus, such sequences are transcribed into RNA but are then excised and are not translated. The term is also used for the excised RNA sequences.

[0154] The "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of different 3' non-coding sequences is exemplified by Ingelbrecht, I. L., et al. (1989) Plant Cell 1:671-680.

[0155] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989. Transformation methods are well known to those skilled in the art and are described below.

[0156] "PCR" or "Polymerase Chain Reaction" is a technique for the synthesis of large quantities of specific DNA segments, consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.). Typically, the double stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a cycle.

[0157] "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including nuclear and organellar genomes, resulting in genetically stable inheritance.

[0158] In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance.

[0159] Host organisms comprising the transformed nucleic acid fragments are referred to as "transgenic" organisms.

[0160] The term "amplified" means the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

[0161] The term "chromosomal location" includes reference to a length of a chromosome which may be measured by reference to the linear segment of DNA which it comprises. The chromosomal location can be defined by reference to two unique DNA sequences, i.e., markers.

[0162] The term "marker" includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A "polymorphic marker" includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes in that pair to be followed. A genotype may be defined by use of one or a plurality of markers.

[0163] The present invention includes, inter alia, compositions and methods for modulating (i.e., increasing or decreasing) the level of plastidic polypeptides described herein in plants. The size of the starch and soluble carbohydrate pools in soybean seeds can be modulated by altering the expression of a specific gene, encoding plastidic phosphoglucomutase (pPGM), which is involved in the starch and soluble carbohydrate biosynthetic pathway.

[0164] Silencing of pPGM gene expression in transgenic soybeans seeds resulted in a drastic decrease of the transient starch pool accompanied by a reduction in soluble carbohydrates and a concomitant increase in combined oil and protein content. Elimination of the transient starch pool by silencing plastidic PGM gene expression in soybean seeds diverted carbon away from the soluble carbohydrate components of dry soybean seeds with the major decrease occurring in the sucrose pool. This is in contrast to PGM mutants of pea, Arabidopsis, and Nicotiana, where the carbon is mainly funneled into the soluble carbohydrate pool.

[0165] The data discussed below further indicates that soybean seeds deficient in plastidic PGM reallocate the carbon destined for starch biosynthesis toward the biosynthesis oil and protein and also alters the sucrose to raffinose family oligosaccharide ratio.

[0166] In contrast, a 40% reduction in storage lipid content was observed in the Arabidopsis mutant pgm-1, which contains a point mutation in the AtPGM gene rendering the polypeptide nonfunctional (Periappuram et al., (2000) Plant Physiol. 122:1193-1199).

[0167] An alteration of plastidic PGM activity affects the allocation of carbon to the soluble carbohydrate pool as well as the allocation of carbon to oil and protein biosynthesis. This is accomplished with no adverse effect on plant and seed phenotype. Since all known soybean cultivars contain transient starch, silencing the plastidic PGM gene in any soybean cultivar should result in a decrease of the transient carbon reserve together with an increase in the combined oil and protein level. This increase in the combined oil and protein level is in addition to the oil and protein levels of the wild-type genotype, regardless of whether the level is low or high. For example, a soybean variety such as Sakaii-18 has 56% of its seed dry weight as protein and 14% as oil. A Pioneer soybean variety, 9306, contains 41% protein and 23% oil (as seed dry weight). Both genotypes would be expected to have an increase in the combined oil and protein content as well as altered sucrose to raffinose family oligosaccharide ratio.

[0168] In one embodiment, the present invention concerns an isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide comprising at least 560 amino acids, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:8 have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, (b) a second nucleotide sequence encoding a second polypeptide comprising at least 560 amino acids, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, or (c) the complement of the first or second nucleotide sequence, wherein the complement and the first or second nucleotide sequence contain the same number of nucleotides and are 100% complementary. The first polypeptide preferably comprises the amino acid sequence of SEQ ID NO:8, and the second polypeptide preferably comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10. The first nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:7, the second nucleotide sequence preferably comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:9. The first and second polypeptides preferably have phosphoglucomutase activity.

[0169] In another embodiment, the present invention relates to a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence.

[0170] In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer.

[0171] In another embodiment, the present invention relates to a vector comprising any of the isolated polynucleotides of the present invention.

[0172] In another embodiment, the present invention relates to an isolated polynucleotide fragment comprising a nucleotide sequence comprised by any of the polynucleotides of the present invention, wherein the nucleotide sequence contains at least 30, 40, 60, 100, 200, 300, 400, 500 or 541 nucleotides.

[0173] In another embodiment, the present invention relates to a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention, and the cell transformed by this method. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0174] In another embodiment, the present invention relates to a method for transforming a cell, comprising transforming a cell with a polynucleotide of the present invention.

[0175] In another embodiment, the present invention relates to a method for producing a transgenic plant comprising transforming a plant cell with any of the isolated polynucleotides of the present invention and regenerating a transgenic plant from the transformed plant cell.

[0176] In another embodiment, a cell, plant, or seed comprising a recombinant DNA construct of the present invention.

[0177] In another embodiment, the present invention concerns an isolated polypeptide comprising: (a) a first amino acid sequence comprising at least 560 amino acids, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:8 have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment, and (b) a second amino acid sequence comprising at least 560 amino acids, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10 have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, based on the Clustal V method of alignment. The first amino acid sequence preferably comprises the amino acid sequence of SEQ ID NO:8, and the second amino acid sequence preferably comprises the amino acid sequence SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:10. The polypeptide preferably has phosphoglucomutase activity.

[0178] In another embodiment, the present invention relates to a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the present invention or any of the recombinant DNA constructs of the present invention.

[0179] In another embodiment, the invention relates to a method of selecting an isolated polynucleotide that alters, i.e., increases or decreases, the level of expression of a phosphoglucomutase gene, protein or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; (b) introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; (c) measuring the level of the phosphoglucomutase RNA, protein or enzyme activity in the host cell containing the isolated polynucleotide or recombinant DNA construct; (d) comparing the level of the phosphoglucomutase RNA, protein or enzyme activity in the host cell containing the isolated polynucleotide or recombinant DNA construct with the level of the phosphoglucomutase RNA, protein or enzyme activity in a host cell that does not contain the isolated polynucleotide or recombinant DNA construct, and selecting the isolated polynucleotide or recombinant DNA construct that alters, i.e., increases or decreases, the level of expression of the phosphoglucomutase gene, protein or enzyme activity in the plant cell.

[0180] In another embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a phosphoglucomutase protein, preferably a plant phosphoglucomutase protein, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, more preferably at least 60 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, and 9, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a phosphoglucomutase protein amino acid sequence.

[0181] In another embodiment, the invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a phosphoglucomutase protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

[0182] In another embodiment, the invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the phosphoglucomutase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

[0183] In another embodiment, this invention concerns a method for suppressing the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity in a transgenic plant, wherein the method comprises: [0184] (a) transforming a plant cell with a fragment of the isolated polynucleotide of the invention; [0185] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0186] (c) selecting a transgenic plant wherein the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity has been suppressed.

[0187] Preferably, the gene encodes a plastidic polypeptide having phosphoglucomutase activity, and the plant is a soybean plant.

[0188] In another embodiment, the invention concerns a recombinant DNA construct comprising: (a) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or (b) the complement of (a); wherein (a) or (b) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant.

[0189] In another embodiment, the invention concerns a method for producing transgenic seed, the method comprising: [0190] (a) transforming a plant cell with the recombinant DNA construct of [0191] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0192] (ii) the complement of (i); [0193] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0194] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0195] (c) selecting a transgenic plant that produces a transgenic seed having an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% as compared to seed obtained from a non-transgenic plant. Preferably, the seed is a soybean seed.

[0196] In another embodiment, the invention concerns a method method for producing transgenic seed, the method comprising: [0197] (a) transforming a plant cell with a recombinant DNA construct comprising [0198] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0199] (ii) the complement of (i); [0200] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0201] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0202] (c) selecting a transgenic plant that produces a transgenic seed having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to seed obtained from a non-transgenic plant.

[0203] Preferably, the transgenic seed differs from an untransformed seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%. Preferably, the seed is a soybean seed.

[0204] In another embodiment, the invention concerns a method for producing defatted meal from transgenic seed, comprising: [0205] (a) transforming a plant cell with a recombinant DNA construct comprising (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0206] (ii) the complement of (i); [0207] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0208] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0209] (c) selecting a transgenic plant that produces a transgenic seed wherein said seed is processed into defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% as compared to defatted meal obtained from seed of a non-transgenic plant.

[0210] Preferably, the defatted meal of the transgenic seed differs from the defatted meal of an untransformed seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the seed is a soybean seed.

[0211] In a another embodiment, the invention concerns a transgenic seed that differs from an non-transgenic seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%, and a decrease in the sucrose content of at least 25%. Preferably, the seed is a soybean seed.

[0212] In another embodiment, the invention concerns a transgenic seed that differs from non-transgenic seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the transgenic seed differs from an untransformed seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%. Preferably, the seed is a soybean seed.

[0213] In another embodiment, the invention concerns a transgenic seed comprising a recombinant construct comprising (i) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; or [0214] (ii) the complement of (i); [0215] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant; [0216] further wherein said transgenic seed is processed to make defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% when compared to defatted meal obtained from a non-transgenic seed.

[0217] Preferably, the defatted meal of the transgenic seed differs from the defatted meal of an untransformed seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the seed is a soybean seed.

[0218] Soybeans can be processed into a number of products. For example, "soy protein products" can include, and are not limited to, those items listed in Table A. "Soy protein products".

TABLE-US-00002 TABLE A Soy Protein Products Derived from Soybean Seeds.sup.a Whole Soybean Products Roasted Soybeans Baked Soybeans Soy Sprouts Soy Milk Specialty Soy Foods/Ingredients Soy Milk Tofu Tempeh Miso Soy Sauce Hydrolyzed Vegetable Protein Whipping Protein Processed Soy Protein Products Full Fat and Defatted Flours Soy Grits Soy Hypocotyls Soybean Meal Soy Milk Soy Protein Isolates Soy Protein Concentrates Textured Soy Proteins Textured Flours and Concentrates Textured Concentrates Textured Isolates .sup.aSee Soy Protein Products: Characteristics, Nutritional Aspects and Utilization (1987). Soy Protein Council.

[0219] "Processing" refers to any physical and chemical methods used to obtain the products listed in Table A and includes, and is not limited to, heat conditioning, flaking and grinding, extrusion, solvent extraction, or aqueous soaking and extraction of whole or partial seeds. Furthermore, "processing" includes the methods used to concentrate and isolate soy protein from whole or partial seeds, as well as the various traditional Oriental methods in preparing fermented soy food products. Trading Standards and Specifications have been established for many of these products (see National Oilseed Processors Association Yearbook and Trading Rules 1991-1992).

[0220] "White" flakes refer to flaked, dehulled cotyledons that have been defatted and treated with controlled moist heat to have a PDI (ASCS: ba10-65) of about 85 to 90. This term can also refer to a flour with a similar PDI that has been ground to pass through a No. 100 U.S. Standard Screen size.

[0221] "Grits" refer to defatted, dehulled cotyledons having a U.S. Standard screen size of between No. 10 and 80.

[0222] "Soy Protein Concentrates" refer to those products produced from dehulled, defatted soybeans by three basic processes: acid leaching (at about pH 4.5), extraction with alcohol (about 55-80%), and denaturing the protein with moist heat prior to extraction with water. Conditions typically used to prepare soy protein concentrates have been described by Pass ((1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New Protein Foods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10, Seed Storage Proteins, pp 302-338).

[0223] "Extrusion" refers to processes whereby material (grits, flour or concentrate) is passed through a jacketed auger using high pressures and temperatures as a means of altering the texture of the material. "Texturing" and "structuring" refer to extrusion processes used to modify the physical characteristics of the material. The characteristics of these processes, including thermoplastic extrusion, have been described previously (Atkinson (1970) U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods, ed. by Altschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367-414). Moreover, conditions used during extrusion processing of complex foodstuff mixtures that include soy protein products have been described previously (Rokey (1983) Feed Manufacturing Technology III, 222-237; McCulloch, U.S. Pat. No. 4,454,804).

[0224] In another embodiment, this invention concerns a method for suppressing the level of expression of a gene encoding a polypeptide having ADP-glucose pyrophosphorylase activity in a transgenic plant, wherein the method comprises: [0225] (a) transforming a plant cell with a fragment SEQ ID NOs:24, 28 or 30, or their complement; [0226] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0227] (c) selecting a transgenic plant wherein the level of expression of a gene encoding a polypeptide having ADP-glucose pyrophosphorylase activity has been suppressed.

[0228] Preferably, the plant is a soybean plant.

[0229] In another embodiment, the invention concerns a recombinant DNA construct comprising: (a) all or part of the nucleotide sequence set forth in SEQ ID NOs:24, 28 or 30; or (b) the complement of (a); wherein (a) or (b) is useful in co-suppression or antisense suppression of endogenous ADP-glucose pyrophosphorylase activity in a transgenic plant.

[0230] In another embodiment, the invention concerns a method for producing transgenic seed, the method comprising: [0231] (a) transforming a plant cell with the recombinant DNA construct of: [0232] (i) all or part of the nucleotide sequence set forth in SEQ ID NO:24, 28 or 30; or [0233] (ii) the complement of (i); [0234] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous ADP-glucose pyrophosphorylase activity in a transgenic plant; [0235] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0236] (c) selecting a transgenic plant that produces a transgenic seed having an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% as compared to seed obtained from a non-transgenic plant.

[0237] Preferably, the seed is a soybean seed.

[0238] In another embodiment, the invention concerns a method for producing transgenic seed, the method comprising: [0239] (a) transforming a plant cell with a recombinant DNA construct comprising [0240] (i) all or part of the nucleotide sequence set forth in SEQ ID NOs:24, 28 or 30; or [0241] (ii) the complement of (i); [0242] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous ADP-glucose pyrophosphorylase activity in a transgenic plant; [0243] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0244] (c) selecting a transgenic plant that produces a transgenic seed having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to seed obtained from a non-transgenic plant.

[0245] Preferably, the transgenic seed differs from an untransformed seed by having an increase in the combined oil and protein content of at least 1.6%, 1.8% or 2.0%. Preferably, the seed is a soybean seed.

[0246] In another embodiment, the invention concerns a method for producing defatted meal from transgenic seed, comprising: [0247] (a) transforming a plant cell with a recombinant DNA construct comprising [0248] (i) all or part of the nucleotide sequence set forth in SEQ ID NOs:24, 28 or 30; or [0249] (ii) the complement of (i); [0250] wherein (i) or (ii) is useful in co-suppression or antisense suppression of endogenous ADP-glucose pyrophosphorylase activity in a transgenic plant; [0251] (b) regenerating a transgenic plant from the transformed plant cell of (a); and [0252] (c) selecting a transgenic plant that produces a transgenic seed wherein said seed is processed into defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% as compared to defatted meal obtained from seed of a non-transgenic plant.

[0253] Preferably, the defatted meal of the transgenic seed differs from the defatted meal of an untransformed seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less. Preferably, the seed is a soybean seed.

[0254] In a another embodiment, the invention concerns a transgenic seed produced by any of the above methods. Preferably, the seed is a soybean seed.

[0255] Regulatory sequences may include, and are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0256] "Tissue-specific" promoters direct RNA production preferentially in particular types of cells or tissues. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (Biochemistry of Plants 15:1-82 (1989)). It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0257] A number of promoters can be used to practice the present invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-specific (preferred), inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0258] In choosing a promoter to use in the methods of the invention, it may be desirable to use a tissue-specific or developmentally regulated promoter. A tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in particular cells/tissues of a plant. Any identifiable promoter may be used in the methods of the present invention which causes the desired temporal and spatial expression.

[0259] Promoters which are seed or embryo specific and may be useful in the invention include patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470; Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z- L, et al. (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barley endosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J. 6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., et al. (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants. Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J. 6:3559-3564 (1987)).

[0260] A plethora of promoters is described in WO 00/18963, published on Apr. 6, 2000, the disclosure of which is hereby incorporated by reference. Examples of seed-specific promoters include, and are not limited to, the promoter for soybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)) .beta.-conglycinin (Chen et al., Dev. Genet. 10:112-122 (1989)), the napin promoter, and the phaseolin promoter.

[0261] In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a cognate gene of a polynucleotide of the present invention so as to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth so as to alter the total concentration and/or alter the composition of the polypeptides of the present invention in plant cell. Thus, the present invention includes compositions, and methods for making, heterologous promoters and/or enhancers operably linked to a native, endogenous (i.e., non-heterologous) form of a polynucleotide of the present invention.

[0262] An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg, Mol. Cell. Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987)). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994). A vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. in Enzymol. 153:253-277 (1987).

[0263] If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.

[0264] Preferred recombinant DNA constructs include the following combinations: a) a nucleic acid fragment corresponding to a promoter operably linked to at least one nucleic acid fragment encoding a selectable marker, followed by a nucleic acid fragment corresponding to a terminator, b) a nucleic acid fragment corresponding to a promoter operably linked to a nucleic acid fragment capable of producing a stem-loop structure, and followed by a nucleic acid fragment corresponding to a terminator, and c) any combination of a) and b) above. Preferably, in the stem-loop structure at least one nucleic acid fragment that is capable of suppressing expression of a native gene comprises the "loop" and is surrounded by nucleic acid fragments capable of producing a stem.

[0265] Preferred methods for transforming dicots and obtaining transgenic plants have been published, among others, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al. (1996) Plant Cell Rep. 15:653-657, McKently et al. (1995) Plant Cell Rep. 14:699-703); papaya (Ling, K. et al. (1991) Bio/technology 9:752-758); and pea (Grant et al. (1995) Plant Cell Rep. 15:254-258). For a review of other commonly used methods of plant transformation see Newell, C. A. (2000) Mol. Biotechnol. 16:53-65. One of these methods of transformation uses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F. (1987) Microbiol. Sci. 4:24-28). Transformation of soybeans using direct delivery of DNA has been published using PEG fusion (PCT publication WO 92/17598), electroporation (Chowrira, G. M. et al. (1995) Mol. Biotechnol. 3:17-23; Christou, P. et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966), microinjection, or particle bombardment (McCabe, D. E. et. Al. (1988) Bio/Technology 6:923; Christou et al. (1988) Plant Physiol. 87:671-674).

[0266] There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated. The regeneration, development and cultivation of plants from single plant protoplast transformants or from various transformed explants are well known in the art (Weissbach and Weissbach, (1988) In.: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc., San Diego, Calif.). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil. The regenerated plants may be self-pollinated. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide(s) is cultivated using methods well known to one skilled in the art.

[0267] In addition to the above discussed procedures, practitioners are familiar with the standard resource materials which describe specific conditions and procedures for the construction, manipulation and isolation of macromolecules (e.g., DNA molecules, plasmids, etc.), generation of recombinant DNA fragments and recombinant expression constructs and the screening and isolating of clones, (see for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press; Maliga et al. (1995) Methods in Plant Molecular Biology, Cold Spring Harbor Press; Birren et al. (1998) Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, N.Y.; Birren et al. (1998) Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y.; Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, N.Y. (1997)).

[0268] Assays to detect proteins may be performed by SDS-polyacrylamide gel electrophoresis or immunological assays. Assays to detect levels of substrates or products of enzymes may be performed using gas chromatography or liquid chromatography for separation and UV or visible spectrometry or mass spectrometry for detection, or the like. Determining the levels of mRNA of the enzyme of interest may be accomplished using northern-blotting or RT-PCR techniques. Once plants have been regenerated, and progeny plants homozygous for the transgene have been obtained, plants will have a stable phenotype that will be observed in similar seeds in later generations.

[0269] In another aspect, this invention includes a polynucleotide of this invention or a functionally equivalent subfragment thereof useful in antisense inhibition or cosuppression of expression of nucleic acid sequences encoding proteins having plastidic phosphoglucomutase activity, most preferably in antisense inhibition or cosuppression of an endogenous plastidic phosphoglucomutase gene.

[0270] Protocols for antisense inhibition or co-suppression are well known to those skilled in the art.

[0271] Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000) Nature 404:804-808). Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998). Recent work has described the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication WO 99/53050 published on Oct. 21, 1999). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression see Wesley, S. V. et al. (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286. A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (WO 99/61632 published on Dec. 2, 1999). The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (WO 02/00894 published Jan. 3, 2002). Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication WO 02/00904, published 3 Jan. 2002.

[0272] The sequences of the polynucleotide fragments used for suppression do not have to be 100% identical to the sequences of the polynucleotide fragment found in the gene to be suppressed. For example, suppression of all the subunits of the soybean seed storage protein .beta.-conglycinin has been accomplished using a polynucleotide derived from a portion of the gene encoding the .alpha. subunit (U.S. Pat. No. 6,362,399). .beta.-conglycinin is a heterogeneous glycoprotein composed of varying combinations of three highly negatively charged subunits identified as .alpha., .alpha.' and .beta.. The polynucleotide sequences encoding the .alpha. and .alpha.' subunits are 85% identical to each other while the polynucleotide sequences encoding the .beta. subunit are 75 to 80% identical to the .alpha. and .alpha.' subunits, respectively. Thus, polynucleotides that are at least 75% identical to a region of the polynucleotide that is target for suppression have been shown to be effective in suppressing the desired target. The polynucleotide may be at least 80% identical, at least 90% identical, at least 95% identical, or about 100% identical to the desired target sequence.

[0273] The isolated nucleic acids and proteins and any embodiments of the present invention can be used over a broad range of plant types, particularly dicots such as the species of the genus Glycine.

[0274] It is believed that the nucleic acids and proteins and any embodiments of the present invention can be with monocots as well including, but not limited to, Graminiae including Sorghum bicolor and Zea mays.

[0275] The isolated nucleic acid and proteins of the present invention can also be used in species from the following dicot genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Antirrhinum, Pelargonium, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Glycine, Pisum, Phaseolus, and from the following monocot genera: Bromus, Asparagus, Hemerocallis, Panicum, Pennisetum, Lolium, Oryza, Avena, Hordeum, Secale, Triticum, Bambusa, Dendrocalamus, and Melocanna.

EXAMPLES

[0276] The present invention is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1

Composition of cDNA Libraries

Isolation and Sequencing of cDNA Clones

[0277] cDNA libraries representing mRNAs from various cattail, corn, rice and soybean tissues were prepared. The characteristics of the libraries are described below.

TABLE-US-00003 TABLE 2 cDNA Libraries from Cattail, Corn, Rice and Soybean Library Tissue Clone etr1c Cattail (Typha latifolia) root etr1c.pk005.f8 p0075 Corn, root/leaf material from dark-grown 7 p0075.cslaf22f day old Seedlings p0075.cslaf22rb p0128 Corn, pooled primary and secondary p0128.cpicz81r immature ear rdi1c Rice (Oryza sativa, Nipponbare) developing rdi1c.pk001.a22 inflorescence at mitotic stage rth1c Rice leaf inoculated with Magnaporta grisea rth1c.pk009.k14f sdp3c Soybean developing pods 8-9 mm sdp3c.pk003.e22 ses4d Soybean embryogenic suspension 4 days ses4d.pk0018.d10 after subculture sdp2c Soybean developing pods 6-7 mm sdp2c.pk008.m2 ssm Soybean shoot meristem ssm.pk0072.e7 ssl Soybean seedling 5-10 day ssl.pk0021.h3 sgs4c Soybean seeds 2 days after germination sgs4c.pk005.b10

[0278] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2

Identification of cDNA Clones

[0279] cDNA clones encoding plastidic phosphoglucomutase proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.

Example 3

Characterization of cDNA Clones Encoding Plastidic Phosphoglucomutase Proteins

[0280] The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to plastidic phosphoglucomutase from Brassica napus (NCBI General Identifier No. 6272125) and Pisum sativum (NCBI General Identifier No. 6272283 and NCBI General Identifier No. 10190529). Shown in Table 3 are the BLAST results for individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs ("Contig"), contigs assembled from an FIS and one or more ESTs ("Contig*"), or sequences encoding the entire protein derived from an FIS, a contig, an EST and PCR, or an FIS and PCR ("CGS"):

TABLE-US-00004 TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to Brassica napus and Pisum sativum Plastidic Phosphoglucomutase Clone Status BLAST pLog Score etr1c.pk005.f8 (FIS) CGS >254.00 (GI No. 6272125; B. napus) Contig Composed of: CGS >254.00 (GI No. 6272283; P. sativum) p0075.cslaf22f (EST) p0075.cslaf22rb (EST) p0128.cpicz81r (EST) rth1c.pk009.k14f EST 58.00 (GI No. 6272283; P. sativum) (EST) sdp3c.pk003.e22 (EST CGS >254.00 (GI No. 6272283; P. sativum) and PCR Fragments) rdi1c.pk001.a22 (FIS) CGS 180.00 (GI No. 10190529; P. sativum)

[0281] The data in Table 4 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8, and 10, and the Brassica napus and Pisum sativum sequences.

TABLE-US-00005 TABLE 4 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Brassica napus and Pisum sativum Plastidic Phosphoglucomutase SEQ ID NO. Percent Identity to 2 79% (GI No. 6272125; B. napus) 4 77% (GI No. 6272283; P. sativum) 6 80% (GI No. 6272283; P. sativum) 8 90% (GI No. 6272283; P. sativum) 10 76% (GI No. 10190529; P. sativum)

[0282] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments, BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a plastidic phosphoglucomutase.

Example 4

Expression of Chimeric Genes in Monocot Cells

[0283] A chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15.degree. C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue.TM.; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3' region.

[0284] The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27.degree. C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0285] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0286] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 .mu.m in diameter) are coated with DNA using the following technique. Ten .mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M solution) and spermidine free base (20 .mu.L of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 .mu.L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the DNA-coated gold particles can be placed in the center of a Kapton.TM. flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic.TM. PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0287] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0288] Seven days after bombardment the tissue can be transferred to N6 medium that contains glufosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing glufosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0289] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 5

Expression of Chimeric Genes in Dicot Cells

[0290] A seed-specific construct composed of the promoter and transcription terminator from the gene encoding the subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin construct includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire construct is flanked by Hind III sites.

[0291] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed construct.

[0292] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26.degree. C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0293] Soybean embryogenic suspension cultures can be maintained in 35 mL of liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0294] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0295] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed construct comprising the phaseolin 5' region, the fragment encoding the instant polypeptides and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0296] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 .mu.L 70% ethanol and resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five .mu.L of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0297] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60.times.15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches of mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0298] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 6

Expression of Chimeric Genes in Microbial Cells

[0299] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.

[0300] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG.TM. low melting agarose gel (FMC). Buffer and agarose contain 10 .mu.g/mL ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase.TM. (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 .mu.L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16.degree. C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 .mu.g/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0301] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25.degree. C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-.beta.-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25.degree. C. Cells are then harvested by centrifugation and re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One .mu.g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 7

Transformation of Somatic Soybean Embryo Cultures

[0302] Generic Stable Soybean Transformation Protocol:

[0303] Soybean embryogenic suspension cultures are maintained in 35 ml liquid media (SB55 or SBP6) on a rotary shaker, 150 rpm, at 28.degree. C. with mixed fluorescent and incandescent lights on a 16:8 h day/night schedule. Cultures are subcultured every four weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.

TABLE-US-00006 TABLE 5 Stock Solutions (g/L): MS Sulfate 100X Stock MgSO.sub.4 7H.sub.2O 37.0 MnSO.sub.4 H.sub.2O 1.69 ZnSO.sub.4 7H.sub.2O 0.86 CuSO.sub.4 5H.sub.2O 0.0025 MS Halides 100X Stock CaCl.sub.2 2H.sub.2O 44.0 KI 0.083 CoCl.sub.2 6H.sub.20 0.00125 KH.sub.2PO.sub.4 17.0 H.sub.3BO.sub.3 0.62 Na.sub.2MoO.sub.4 2H.sub.2O 0.025 MS FeEDTA 100X Stock Na.sub.2EDTA 3.724 FeSO.sub.4 7H.sub.2O 2.784 B5 Vitamin Stock 10 g m-inositol 100 mg nicotinic acid 100 mg pyridoxine HCl 1 g thiamine SB55 (per Liter, pH 5.7) 10 ml each MS stocks 1 ml B5 Vitamin stock 0.8 g NH.sub.4NO.sub.3 3.033 g KNO.sub.3 1 ml 2,4-D (10 mg/mL stock) 60 g sucrose 0.667 g asparagine SBP6 same as SB55 except 0.5 ml 2,4-D SB103 (per Liter, pH 5.7) 1X MS Salts 6% maltose 750 mg MgCl.sub.2 0.2% Gelrite SB71-1 (per Liter, pH 5.7) 1X B5 salts 1 ml B5 vitamin stock 3% sucrose 750 mg MgCl.sub.2 0.2% Gelrite

[0304] Soybean embryogenic suspension cultures are transformed with plasmid DNA by the method of particle gun bombardment (Klein et al (1987) Nature 327:70). A DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit) is used for these transformations.

[0305] To 50 ml of a 60 mg/ml 1 .mu.m gold particle suspension is added (in order); 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l spermidine (0.1 M), and 50 .mu.l CaCl.sub.2 (2.5 M). The particle preparation is agitated for 3 min, spun in a microfuge for 10 sec and the supernatant removed. The DNA-coated particles are then washed once in 400 .mu.l 70% ethanol and re suspended in 40 .mu.l of anhydrous ethanol. The DNA/particle suspension is sonicated three times for 1 sec each. Five .mu.l of the DNA-coated gold particles are then loaded on each macro carrier disk. For selection, a plasmid conferring resistance to hygromycin phosphotransferase (HPT) may be co-bombarded with the silencing construct of interest.

[0306] Approximately 300-400 mg of a four week old suspension culture is placed in an empty 60.times.15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1000 psi and the chamber is evacuated to a vacuum of 28 inches of mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue is placed back into liquid and cultured as described above.

[0307] Eleven days post bombardment, the liquid media is exchanged with fresh SB55 containing 50 mg/ml hygromycin. The selective media is refreshed weekly. Seven weeks post bombardment, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Thus each new line is treated as an independent transformation event. These suspensions can then be maintained as suspensions of embryos maintained in an immature developmental stage or regenerated into whole plants by maturation and germination of individual somatic embryos.

[0308] Independent lines of transformed embryogenic clusters are removed from liquid culture and placed on a solid agar media (SB103) containing no hormones or antibiotics. Embryos are cultured for four weeks at 26.degree. C. with mixed fluorescent and incandescent lights on a 16:8 h day/night schedule. During this period, individual embryos are removed from the clusters and screened for alterations in gene expression.

[0309] It should be noted that any detectable phenotype, resulting from the co-suppression of a target gene, can be screened at this stage. This would include, but not be limited to, alterations in protein content, carbohydrate content, growth rate, viability, or the ability to develop normally into a soybean plant.

Example 8

Plasmid DNAs for "Complementary Region" Co-Suppression

[0310] The plasmids used in these experiments were made using standard cloning methods well known to those skilled in the art (Sambrook et al (1989) Molecular Cloning, CSHL Press, New York). A starting plasmid pKS18HH (U.S. Pat. No. 5,846,784 the contents of which are hereby incorporated by reference) contains a hygromycin B phosphotransferase (HPT) obtained from E. coli strain W677 under the control of a T7 promoter and the 35S cauliflower mosaic virus promoter. Plasmid pKS18HH thus contains the T7 promoter/HPT/T7 terminator cassette for expression of the HPT enzyme in certain strains of E. coli such as NovaBlue(DE3) [from Novagen], that are lysogenic for lambda DE3 (which carries the T7 RNA Polymerase gene under lacV5 control). Plasmid pKS18HH also contains the 35S/HPT/NOS cassette for constitutive expression of the HPT enzyme in plants, such as soybean. These two expression systems allow selection for growth in the presence of hygromycin to be used as a means of identifying cells that contain the plasmid in both bacterial and plant systems. pKS18HH also contains three unique restriction endonuclease sites suitable for the cloning other chimeric genes into this vector. Plasmid ZBL100 (PCT Application No. WO 00/11176 published on Mar. 2, 2000) is a derivative of pKS18HH with a reduced NOS 3' terminator. Plasmid pKS67 is a ZBL100 derivative with the insertion of a beta-conglycinin promoter, in front of a NotI cloning site, followed by a phaseolin 3' terminator (described in PCT Application No. WO 94/11516, published on May 26, 1994).

[0311] The 2.5 kb plasmid pKS17 contains pSP72 (obtained from Promega Biosystems) and the T7 promoter/HPT/T7 3' terminator region, and is the original vector into which the 3.2 kb BamHI-SalI fragment containing the 35S/HPT/NOS cassette was cloned to form pKS18HH. The plasmid pKS102 is a pKS17 derivative that is digested with XhoI and SalI, treated with mung-bean nuclease to generate blunt ends, and ligated to insert the following linker:

TABLE-US-00007 SEQ ID NO: 18 GGCGCGCCAAGCTTGGATCCGTCGACGGCGCGCC

[0312] The plasmid pKS83 has the 2.3 kb BamHI fragment of ML70 containing the Kti3 promoter/NotI/Kti3 3' terminator region (described in PCT Application No. WO 94/11516, published on May 26, 1994) ligated into the BamHI site of pKS17.

Example 9

Suppression by ELVISLIVES Complementary Region

[0313] Constructs have now been made which have "synthetic complementary regions" (SCR). In this example the target sequence is placed between complementary sequences that are not known to be part of any biologically derived gene or genome (i.e. sequences that are "synthetic" or conjured up from the mind of the inventor). The target DNA would therefore be in the sense or antisense orientation and the complementary RNA would be unrelated to any known nucleic acid sequence. It is possible to design a standard "suppression vector" into which pieces of any target gene for suppression could be dropped. The plasmids pKS106, pKS124, and pKS133 (SEQ ID NO:17) exemplify this. One skilled in the art will appreciate that all of the plasmid vectors contain antibiotic selection genes such as, but not limited to, hygromycin phosphotransferase with promoters such as the T7 inducible promoter.

[0314] pKS106 uses the beta-conglycinin promoter while the pKS124 and pKS133 plasmids use the Kti promoter, both of these promoters exhibit strong tissue specific expression in the seeds of soybean. pKS106 uses a 3' termination region from the phaseolin gene, and pKS124 and pKS133 use a Kti 3' termination region. pKS106 and pKS124 have single copies of the 36 nucleotide EagI-ELVISLIVES sequence surrounding a NotI site (the amino acids given in parentheses are back-translated from the complementary strand): SEQ ID NO:19

[0315] EagI E L V I S L I V E S NotI

[0316] CGGCCG GAG CTG GTC ATC TCG CTC ATC GTC GAG TCG GCGGCCGC

[0317] (S) (E) (V) (I) (L) (S) (I) (V) (L) (E) EagI [0318] CGA CTC GAC GAT GAG CGA GAT GAC CAG CTC CGGCCG pKS133 has 2.times. copies of ELVISLIVES surrounding the NotI site: SEQ ID NO:20

[0319] EagI E L V I S L I V E S EagI E L V I S

[0320] cggccggagctggtcatctcgctcatcgtcgagtcg gcggccg gagctggtcatctcg

[0321] L I V E S NotI (S)(E (V)(I)(L)(S)(I)(V)(L)(E) EagI

[0322] ctcatcgtcgagtcg gcggccgc cgactcgacgatgagcgagatgaccagctc cggccgc

[0323] (S)(E)(V)(I)(L)(S)(I)(V)(L)(E) EagI

[0324] cgactcgacgatgagcgagatgaccagctc cggccg

[0325] The idea is that the single EL linker (SCR) can be duplicated to increase stem lengths in increments of approximately 40 nucleotides. A series of vectors will cover the SCR lengths between 40 bp and the 300 bp. Various target gene lengths are also under evaluation. It is believed that certain combinations of target lengths and complementary region lengths will give optimum suppression of the target, although preliminary results would indicate that the suppression phenomenon works well over a wide range of sizes and sequences. It is also believed that the lengths and ratios providing optimum suppression may vary somewhat given different target sequences and/or complementary regions.

[0326] The plasmid pKS106 is made by putting the EagI fragment of ELVISLIVES (SEQ ID NO:19) into the NotI site of pKS67. The ELVISLIVES fragment is made by PCR using two primers and no other DNA:

TABLE-US-00008 SEQ ID NO:21 5'-GAATTCCGGCCGGAGCTGGTCATCTCGCTCATCGTCGAGTCGGCGG CCGCCGACTCGACGATGAGCGAGATGACCAGCTCCGGCCGGAATTC-3' SEQ ID NO: 22 5'-GAATTCCGGCCGGAG-3'

[0327] The product of the PCR reaction is digested with EagI (5'-CGGCCG-3') and then ligated into NotI digested pKS67. The term "ELVISLIVES" and "EL" are used interchangeably herein.

[0328] Additional plasmids can be used to test this example. For example, pKS121 contains the Kti3 promoter/NotI/Kti3 3' terminator fragment analogous to pKS83 inserted into the BamHI-SalI digested pKS102. The EagI digested ELVISLIVES cloning site made from SEQ ID NOs:14 and 15 is inserted into the NotI site of pKS121 to form pKS124. The EagI digested EL PCR product can be ligated into NotI digested pKS124 to form the 2XEL plasmid, pKS133 (SEQ ID NO:17), containing two copies of ELVISLIVES. An additional 2XEL vector, pKS151, is similar to pKS133 except for the addition of a second hygromycin phosphotransferase gene with a 35S-CaMV promoter. Any synthetic sequence, or naturally occurring sequence, can be used in an analogous manner. The addition of a 574 base pair NotI fragment (SEQ ID NO:14) into a NotI-digested pKS133 produces pTC103. The 574 base pair Not I fragment (SEQ ID NO:14) contains a 541 base pair region (SEQ ID NO:15) of the soybean plastid phosphoglucomutase coding region (SEQ ID NO:8).

Example 10

Down Regulation of Plastidic Phosphoglucomutase in Soybean

[0329] Soybean was transformed with the plasmid DNA, pTC103, and transgenic lines were selected. Transgenic lines were screened for down regulation of plastidic phosphoglucomutase in soybean. The screening assay involved iodine staining for the presence or absence of starch in immature seeds (mid-pod stage). The method involved harvesting half of the seed, and putting that seed on dry ice and storing at -80 C. The other half of the seed was placed in 100% ethanol overnight, and subsequently stained with water:lugol (4:1) solution for 10 to 30 minutes at room temperature. Lugol is an iodine/potassium iodide solution, commercially available from Sigma.

[0330] Four out of nineteen events showed a clear reduction in iodine staining indicating a reduction in starch content. This may reflect a 21% cosuppression success with the hairpin construct. Three additional events showed potential reduction in iodine staining, although the differences in staining were subtle. The segregation patterns of events 100-2-1 and 108-3-1 are consistent with a theoretical segregation of a dominant co-suppression (1:3).

TABLE-US-00009 TABLE 6 Summary of Iodine Screen Sum + Events 4 - Events 12 ? Events 3 Total Events Analyzed 19 Events with no 2 plants/sterile/dwarf Total Events 27

TABLE-US-00010 TABLE 7 Seed segregation information of potential positive pPGM events. Event Plant D:L seed ratio Note 100-2-1 1 1:5 clear positive 100-2-1 2 3:2 clear positive 100-2-1 3 1:5 clear positive 108-3-1 1 0:6 clear positive 108-3-1 2 2:4 clear positive 108-3-1 3 1:5 clear positive 105-2-3 1 4:0 negative 105-2-3 2 1:5 clear positive 105-1-6 1 4:0 negative 105-1-6 3 2:2 clear positive 105-1-1 1 4:2 D/L 105-1-1 2 0:6 D/L 105-1-1 3 6:0 D/L 101-2-6 1 6:0 D/L 101-2-6 3 2:3 D/L 102-3-3 1 2:3 D/L 102-3-3 2 3:0 D/L D = dark blue stain, L = light blue or no stain, D/L in between dark and light stain

Example 11

Silencing of Plastidic Phosphoglucomutase (pPGM) Results in a Stable Reduction of Transient Starch Accumulation in Developing Soybean Seeds

[0331] Transgenic soybean events were produced as described in Example 10. Developing soybean seeds were harvested at approximately mid-maturity (20 to 30 days after flowering (DAF)) and starch content was quantified as described in Brown and Huber, Physiologia Plantarum 72:518-524 (1988). T1 seeds from three transgenic events showed about an 80% reduction in starch content as compared to wild-type seeds (Table 8). The starch data correlated with the iodine staining described in Example 10, i.e., seeds that do not stain blue have a significant reduction in starch content while blue-staining seeds have starch contents similar to wild-type

TABLE-US-00011 TABLE 8 Starch Content of T1 Seeds (Approximately Mid-maturity) Segregating for Co-suppression of pPGM. B = Seeds Staining Blue. W = Seeds Not Staining. Blue Staining Indicates Presence of Starch. Iodine Mg starch/g seed % of wild- Event score mean st. dev. type WT (Jack) B 22.83 5.69 100 108-3-1 W 5.20 2.24 20 108-3-1 B 25.92 -- 100 105-1-8 nd nd nd nd 100-2-1 W 5.27 1.66 22 100-2-1 B 24.27 5.40 100 105-1-6 W 2.69 3.81 14 105-1-6 B 18.87 6.76 100 nd = not determined

[0332] The soluble carbohydrate composition of developing transgenic seeds was measured by high performance anion exchange chromatography/pulsed amperometric detection (HPAE/PAD). Individual seeds from transgenic lines were harvested at mid-maturity (20 to 30 DAF) for detection of carbohydrate composition. The seeds were frozen in liquid nitrogen, ground with a mortar and pestle, and transferred to 15 ml microcentrifuge tubes. Ethanol (80%) was added to the tubes and the samples were heated to 70.degree. C. for 15 minutes. Samples were centrifuged at 4000 rpm for 15 minutes at 4.degree. C. and the supernatant collected. The pellet was re-extracted two additional times with 80% ethanol at 70.degree. C. The supernatants were combined, dried down in a speedvac, and the pellet re-suspended in water. Furthermore, the extracts were filtered through a 0.2 .mu.m Nylon-66 filter (Rainin, Emeryville, Calif.) and analyzed by HPAE/PAD using a DX500 anion exchange analyzer (Dionex, Sunnyvale, Calif.) equipped with a 250.times.4 mm CarboPac PA1 anion exchange column and a 25.times.4 mm CarboPac PA guard column. Soluble carbohydrates were separated with a 30 minute isocratic run of 0.5 mM NaAc in 150 mM NaOH at a flow rate of 1.0 mL/min. A dilution series of glucose, fructose, sucrose, raffinose, stachyose and verbascose were used as external standards.

[0333] Soluble carbohydrate analysis on 6 seeds per event segregating for iodine staining (starch presence) indicated no major change in concentration of sucrose and total soluble carbohydrates at approximately mid-maturity (Table 9). No raffinose family oligosaccharides (RFOs) were observed at this stage of development; hence, no sucrose to RFO ratio could be determined.

TABLE-US-00012 TABLE 9 Sucrose and Total Soluble Sugar Content (mg/g seed) of Segregating T1 Seeds Harvested at Mid-maturity. D = Dark Blue Stain, L = Light Blue or No Stain Plant Seed Sucrose Total Soluble Sugars Event No. Score mean std. mean std. 108-3-1 1 L 22.8 3.1 26.5 3.8 108-3-1 3 D 13.1 -- 17.2 -- 108-3-1 3 L 10.4 5.9 14.7 5.4 105-1-8 nd* nd nd nd nd 100-2-1 2 D 16.0 4.7 21.0 6.0 100-2-1 2 L 18.6 4.7 24.3 5.6 105-1-6 1 D 31.5 12.5 36.4 3.8 105-1-6 3 D 23.1 2.6 27.3 2.6 105-1-6 3 L 18.2 5.2 23.4 5.4 nd* = not determined

[0334] T1 seeds from event 108-3-1 were planted in a growth chamber and T2 seeds were harvested at mid-maturity and screened for starch presence using the iodine screen described in Example 10. Starch and soluble carbohydrate content of T2 seeds were determined as described above.

[0335] T2 seeds in which the pPGM gene is silenced (referred to as PGM-KO) showed an 80% decrease in starch content (Table 10). Null-segregating T2 seeds were also identified which had wild-type starch levels (referred to as PGM-WT). No major differences in soluble carbohydrate concentrations between pPGM-silenced T2 seeds (PGM-KO) and wild-type seeds (Jack or PGM-WT) were observed at this stage of development. This data is similar to data obtained from T1 seeds (Table 8 and Table 9) and suggest that the event is inherited in a stable manner.

TABLE-US-00013 TABLE 10 Starch, Sucrose and Total Soluble Carbohydrate Content of T2 Seeds (Growth Chamber Grown) from pPGM-Silenced Seeds Harvested at Mid-maturity. B = Seeds Staining Blue. W = Seeds Not Staining. Blue Staining Indicates Presence of Starch. mg mg mg Total Starch/g Iodine Sucrose/ CHOs/g Seed % of Name Event Score g Seed Seed (mean) WT Jack Control B 15.9 19.8 22.83 100 PGM-KO 108-3-1 W 14.8 21.3 3.7 22 PGM-WT 108-3-1 B 16.6 19.7 16.89 100

[0336] Starch quantitation of developing soybean seeds harvested at 20, 30, 40 and 50 DAF indicated that throughout seed development, the starch accumulation of a pPGM-silenced soybean was reduced by 85% as compared to its null segregant (FIG. 2).

Example 12

Silencing of Plastidic Phosphoglucomutase Decreases Total Carbohydrate Content and Alters the Sucrose to Raffinose Family Oligosaccharide Ratio in Mature Seeds

[0337] Transgenic T2 soybean seeds were harvested at mid-maturity and screened for reduced starch content using the iodine screen described in Example 10. In total, three thousand seeds (field and growth chamber grown) from 436 plants representing 21 different events were screened. A secondary screen using 20 seeds per plant was conducted to identify potential homozygotes. Five events (108-3-1, 105-1-8, 100-2-1, 105-1-7 and 100-3-2) showed a decreased or no iodine staining.

[0338] Carbohydrate analysis (determined as described in Example 11) of mature T2 seeds from plants grown in a growth chamber (GC) revealed a decrease in total soluble carbohydrates of approximately 11% with sucrose being the major sugar affected (38% decrease) as compared to their null segregant (FIG. 3). Carbohydrate analysis of field grown events revealed a decrease in total soluble carbohydrates of approximately 14 to 25% together with a 30 to 39% decrease in sucrose. In both the growth chamber and field grown plants, mature seeds from the pPGM-silenced events (PGM-KO) were characterized with a distinctive change in sucrose to RFO ratio. Under these conditions the sucrose to RFO ratio of wild-type seeds averaged around 1.3 to 1.7 while the sucrose to RFO ratio of pPGM co-suppressed seeds averaged around 0.7 to 0.9.

[0339] Seeds from a selected number of events were grown in a growth chamber to produce T3 seeds. Carbohydrate analysis of T3 seeds from pPGM-silenced events revealed a decrease in total soluble carbohydrates of approximately 24 to 35% as compared to their wild-type. Sucrose content decreased by 35 to 48% whereas RFO decreased by approximately 14% (FIG. 4).

[0340] The carbohydrate profile of these T3 seeds is thus very similar to R2 seeds and indicates (as was seen with the starch content) that the pPGM trait is inherited in a stable manner. The sucrose to RFO ratio of pPGM co-suppressed R3 seeds (ratio of 1.2 to 1.5) was as compared to the null segregant (ratio of 1.6 to 2.0). Although the sucrose to RFO ratio in T3 seeds is somewhat higher as compared to T2 seeds, the decrease in the sucrose to RFO ratio between pPGM co-suppressed seeds and wild-type seeds averages around 0.5.

Example 13

Silencing of Plastidic Phosphoglucomutase (pPGM) Results in an Increased Accumulation of Oil and/or Protein in Mature Seeds

[0341] Total oil and protein content of mature T2 seeds from event 108-3-1-1 showing a pPGM-silenced phenotype was determined and compared to the oil and protein content of a pPGM null segregant (wild-type phenotype) from the same event (Table 11). Percent oil and percent protein changes were expressed on a seed dry weight basis. Seed composition of wild-type 92B91 was performed as a control reference. Protein and oil were measured by Woodson-Tenant Labs (Des Moines, Iowa), using standard AOAC methods; combustion method for protein (AOAC Official Method 990.03; 2000 AOAC International), and ether extraction method for oil (AOAC Official Method 920.39; 2000 AOAC International).

[0342] Field grown pPGM-silenced seeds showed an increased oil content of up to 2% on a seed dry wt basis and a slightly increased protein content (1% on a seed dry wt basis) as compared to seeds from the null segregant. Total oil content of growth chamber grown pPGM co-suppressed seeds increased by 4% on a seed dry wt basis. This high increase in oil content was accompanied with a decreased protein content (Table 11). No major difference was observed between the seed composition of PGM-WT and the wild-type cultivar 92B91.

TABLE-US-00014 TABLE 11 Seed Composition of T2 seeds from a pPGM-Silenced Line (PGM- KO) and a Null Segregant (PGM-WT) for Event 108-3-1-1 % Oil & Growth % Oil % Protein Protein ID Condition Phenotype % Oil % Protein Increase Increase Increase 2097-7 Growth PGM-KO 23.39 37.08 4.19 -3.04 1.15 Chamber 2097-7 Field PGM-KO 21.22 41.25 2.20 +1.12 3.32 2097-1 Field PGM-WT 19.20 40.12 -- -- -- 92B91 Field wild-type 20.36 40.40 -- -- --

[0343] T2 seeds from event 108-3-1-1 were planted in a growth chamber to produce T3 seeds. This T3 generation of mature seeds from pPGM co-suppressed seeds showed the greatest increase in protein content rather than oil content when compared to their null segregant (Table 12; percent oil and percent protein changes were calculated on a seed dry weight basis). Interestingly, the sum of both increase in oil and protein content was similar for T3 seeds from both plants, ranging from 1.76 to 1.83%, on a seed dry wt basis.

TABLE-US-00015 TABLE 12 Seed Composition of T3 Seeds from Two pPGM-Silenced Plants (PGM-KO) and a Null Segregant (PGM-WT) % Oil & % Oil % Protein Protein Event ID Phenotype % Oil % Protein Increase Increase Increase 108-3-1-1 JS1-261 PGM-KO 22.03 40.09 -0.61 2.37 1.76 108-3-1-1 JS2-265 PGM-KO 22.99 39.21 0.35 1.49 1.83 108-3-1-1 JS3-2642 PGM-WT 22.64 37.72 -- -- --

[0344] T3 seeds from several other events having cv. Jack as their genetic background showed similar trends as observed with the event 108-3-1-1, which has cv. 92B91 as its genetic background. The increase in the sum of oil and protein content ranged from 0.98 to 3.14% as expressed on a seed dry wt basis (Table 13).

TABLE-US-00016 TABLE 13 Seed Composition of T3 Seeds from pPGM-Silenced (PGM-KO) and PGM Wild-Type (PGM-WT) Events in a cv. Jack Background % Oil & % % % Oil % Protein Protein Event Phenotype Oil Protein Increase Increase Increase 101-2-6-3 PGM-WT 22.36 38.48 -- -- -- 105-1-8-2 PGM-KO 21.29 40.53 -1.07 2.05 0.98 100-2-1-1 PGM-KO 23.92 40.06 1.56 1.58 3.14 100-2-1-1 PGM-KO 21.79 40.69 -0.57 2.21 1.64

[0345] Homozygous pPGM-silenced (PGM-KO) and pPGM-wild-type (PGM-WT) seeds, originating from crosses between pPGM knockout event and elite germplasm, were grown in the field in 2003 and in 2004. The seed compositions were determined and are shown in Table 14. The percent oil, percent protein and percent combined oil and protein changes are expressed on a dry weight basis. The moisture content of each sample was measured in the 2004 experiment. For the 2003 data, a moisture content of 13% was assumed, to calculate the dry weight values. For the data shown in Table 14, a range of 1.0% to 3.5% was observed for the percent increase in oil and protein.

TABLE-US-00017 TABLE 14 Seed Composition of Field Grown Homozygous pPGM- Silenced (PGM-KO) and PGM-Wild-Type (PGM-WT) Seeds. % Oil & Elite % Oil % Protein Protein Background Year Phenotype % Oil % Protein Increase Increase Increase 92B63 2003 PGM-KO 22.7 40.6 1.3 -0.3 1.0 92B63 2003 PGM-WT 21.4 40.9 -- -- -- 92B63 2004 PGM-KO 19.1 42.2 1.1 0.5 1.6 92B63 2004 PGM-WT 18.0 41.7 -- -- -- 92B91 2003 PGM-KO 25.7 39.4 2.5 1.0 3.5 92B91 2003 PGM-WT 23.2 38.4 -- -- -- 92B91 2004 PGM-KO 21.4 40.2 1.7 0.3 2.0 92B91 2004 PGM-WT 19.7 39.9 -- -- -- 93B87 2003 PGM-KO 23.1 40.2 0.7 1.2 1.9 93B87 2003 PGM-WT 22.4 39.0 -- -- -- 93B87 2004 PGM-KO 21.5 42.4 1.4 0.8 2.2 93B87 2004 PGM-WT 20.1 41.6 -- -- --

Example 14

Characterization of Defatted Meal from Transgenic Soybean Seeds with Silenced Plastidic Phosphoglucomutase (pPGM)

[0346] T2 soybean seeds were ground to a fine powder and oil was extracted using heptane. Approximately 200 mg of soybean seed were weighed and transferred into pre-weighed 10 ml glass screw cap tubes. Two ml of heptane were added and the mixture was vortexed for 15 min at room temperature. The glass tubes were centrifuged at 3500 rpm for 20 minutes and the heptane was carefully decanted into a new, pre-weighed glass tube. The pellet was re-extracted another three times as described above and all supernatants were pooled. The hexane was removed by evaporation using a speedvac. Total oil content was determined gravimetrically.

[0347] An aliquot of the defatted meal was used for protein determination. About 60 mg of meal was transferred to a 15 ml polypropylene tube and 10 ml 0.1 N NaOH were added and the mixture was incubated at 60.degree. C. for 1 hr. One ml of the suspension was transferred to a 2 ml eppendorf tube and centrifuged at 14,000 rpm for 3 minutes. The supernatant was diluted several fold and the protein content was determined using a microplate assay based on Sigma Total Protein Protocol (Procedure 541-2, Sigma).

[0348] The soluble carbohydrate profile of defatted soybean meal from T2 seeds is shown in FIG. 5. Sucrose to RFO ratios of 0.82 and 0.87 were observed for the pPGM-suppressed event, 108-3-1-1.

[0349] The percent oil and percent protein content of defatted meal from T2 Seeds of event 108-3-1-1 was determined on a wet-weight basis and is presented in Table 15. A percent increase in combined oil and protein of 3.02% and 4.82% was observed for defatted meal from a pPGM-suppressed line grown in the growth chamber and in the field, respectively.

[0350] The percent oil and percent protein content of defatted meal from T2 Seeds of event 108-3-1-1 was determined on a dry-weight basis and is presented in Table 16. A percent increase in combined oil and protein of 3.20% and 5.17% was observed for defatted meal from a pPGM-suppressed line grown in the growth chamber and in the field, respectively.

TABLE-US-00018 TABLE 15 Characterization of Defatted Meal (wet-weight basis) from T2 Seeds of Event 108-3-1-1 from a pPGM Co-suppressed Events (PGM-KO) and a Null Segregant (PGM-WT) % Oil & Growth % Oil % Protein Protein ID Condition Phenotype % Oil % Protein Increase Increase Increase 2097-7 Growth PGM-KO 18.82 37.54 3.40 -0.38 3.02 Chamber 2097-7 Field PGM-KO 17.65 40.51 2.23 2.59 4.82 2097-1 Field PGM-WT 15.42 37.92 -- -- -- 92B91 Field wild-type 16.92 36.80 -- -- --

TABLE-US-00019 TABLE 16 Characterization of Defatted Meal (dry-weight basis) from T2 seeds of Event 108-1-1 from a pPGM Co-suppressed Event (PGM-KO) and a Null Segregant (PGM-WT) % Oil & Growth % Oil % Protein Protein ID Condition Phenotype % Oil % Protein Increase Increase Increase 2097-7 Growth PGM-KO 18.82 40.31 3.40 -0.20 3.20 Chamber 2097-7 Field PGM-KO 17.65 43.45 2.23 2.94 5.17 2097-1 Field PGM-WT 15.42 40.51 -- -- -- 92B91 Field wild-type 16.92 39.35 -- -- --

Example 15

Characterization of cDNA Clones Encoding the Large Subunit and the Small Subunit Polypeptides of Soybean ADP-Glucose Pyrophosphorylase

[0351] A BLAST analysis of soybean sequences identified cDNA clones that encoded proteins with high sequence similarity to the large subunit of ADP-glucose pyrophosphorylase from chickpea, Cicer arietinum (NCBI General Identifier No. 13487785), and to the small subunit, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (NCBI General Identifier No. 29421116). SEQ ID NO:24, encoded by SEQ ID NO:23, corresponds to the amino acid sequence of a large subunit polypeptide of soybean ADP-glucose pyrophosphorylase and has a pLog score of greater than 254 when compared to the large subunit of ADP-glucose pyrophosphorylase from chickpea (SEQ ID NO:25; GI No. 13487785). SEQ ID NO:28, encoded by SEQ ID NO:27, corresponds to the amino acid sequence of a first small subunit polypeptide (SS1) of soybean ADP-glucose pyrophosphorylase and has a pLog score of greater than 254 when compared to the small subunit polypeptide, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (SEQ ID NO:31; GI No. 29421116). SEQ ID NO:30, encoded by SEQ ID NO:29, corresponds to the amino acid sequence of a second small subunit polypeptide (SS2) of soybean ADP-glucose pyrophosphorylase and has a pLog score of greater than 254 when compared to the small subunit polypeptide, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (SEQ ID NO:31; GI No. 29421116).

[0352] The large subunit amino acid sequences of SEQ ID NOs:24, 25 and 26 were aligned and the percent sequence identities were calculated for each pair of sequences. The soybean large subunit amino acid sequence of SEQ ID NO:24 is 79.1% identical to the large subunit of ADP-glucose pyrophosphorylase from chickpea (SEQ ID NO:25; GI No. 13487785), and is 99.6% identical to SEQ ID NO:26 (SEQ ID NO:248406 from U.S. Patent Application US2004031072).

[0353] The small subunit amino acid sequences of SEQ ID NOs:28, 30, 31 and 32 were aligned and the percent sequence identities were calculated for each pair of sequences. The soybean small subunit SS1 amino acid sequence of SEQ ID NO:28 is 94.6% identical to the small subunit polypeptide, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (SEQ ID NO:31; GI No. 29421116), and is 98.3% identical to SEQ ID NO:32 (SEQ ID NO:251944 from U.S. Patent Application US2004031072). The soybean small subunit SS2 amino acid sequence of SEQ ID NO:30 is 94.0% identical to the small subunit polypeptide, PvAGPS1, of ADP-glucose pyrophosphorylase from Phaseolus vulgaris (SEQ ID NO:31; GI No. 29421116), and is 100% identical to SEQ ID NO:32 (SEQ ID NO:251944 from U.S. Patent Application US2004031072). The soybean small subunit SS1 polypeptide (SEQ ID NO:28) and the SS2 polypeptide (SEQ ID NO:30) have 98.3% sequence identity with each other.

[0354] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0355] Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode full-length polypeptides for the large and small subunits of ADP-glucose pyrophosphorylase from soybean.

Sequence CWU 1

1

3212112DNATypha latifolia 1gcaccagctc gttatcgcca cttatcgctc tctcaatctc tctctccata cttgcaagaa 60atggcaatgt cggtgcctac tatgaggttg catcccctcg tcccctcttc gaagcttctc 120tctccctcct cttcgtcgcc ggcggtgctg gtctcttccc ggattcctct cctctctctt 180aggaggccaa acctgaggtt ctccgtcaag gctaccgctt cttccactcc gtccacggcc 240gaaagcataa agatcaagtc gatacccacc aagccagtag aagggcagaa gactgggact 300agcggattaa ggaagaaggt taaggttttc cagcaggaga attacttggc aaactggatt 360caggcactgt ttaattcctt gccgctggag gattacaaga atggattgct ggttttggga 420ggtgatgggc ggtactttaa ccgagaggct gcacagataa tcatcaagat tgctgctgga 480aatggtgttg gaaaaattct tgttggcagg gatggtatca tgtcaactcc tgctgtatct 540gcagtaatac gtaaacagaa ggcaaatggt ggttttatca tgagtgcaag ccataatcct 600ggtggtccgg actatgattg gggcattaag tttaattaca gcagtggaca acctgcacct 660gaatcaatta ctgacaaaat ctacggtaac actctttcga tttctgaaat aaaaatatca 720gatatacctg atattgatct atccagtcta ggtgttacca attatggcaa cttttctgtg 780gaggtggtag accctgtttc agattacttg gagttaatgg agaatgtgtt tgattttcag 840ctcatcaaag gtcttctttc tcgatctgat ttcaggttta catttgatgc gatgcatgca 900gtaacaggtg catatgcaaa acctatcttt gtggaacggc ttcgagctag cccggattgt 960gttttaaatg gagtgcctct tgaagatttt ggccatggtc acccagaccc caatctgacg 1020tatgctaagg agcttgttga tgtaatgtat accacagatg cacctgatct aggagcagca 1080agtgatggtg atggtgatcg aaacatgatt cttggaagac gtttctttgt tacaccatca 1140gattctgttg caatgattgc cgctaatgca caggcggcta ttccttattt ccaagctggt 1200cccaaaggac ttgctaggtc tatgccaaca agcggtgctc ttgatcgtgt agccgaaaaa 1260ttgaaccttc cattctttga ggttccaact ggttggaagt tttttggaaa tctgatggat 1320gctgggaagt tgtccatctg tggggaggaa agttttggca caggttctga tcacatccgg 1380gagaaggatg gcatctgggc tgttttggct tggctttcca taattgcgta cagaaacaag 1440gacaaaaaga ttggagagaa attagtctct gttgaagata ttgctaagga gcactgggca 1500aaatatggca ggaacttctt ttctcgatat gattacgaag aatgcgaatc ggaaggagca 1560aataaaatga tgcagcacct tagggacttt atctcgacaa gcaagcctgg agaacaatat 1620ggaaattata ctcttcaatt ttcagatgac ttttcctaca ctgaccctgt agacggcagt 1680gtagcatcca agcaagggct acgatttgtt ttcacagatg gatcaagggt tatctatcgt 1740ctctcgggta ctggatcggc cggtgcaact atacggatat atgttgaaca attcgagccc 1800gatgtctcca agcatgatgt ggatgcacaa gcagcattaa agcctttgat agacctcgca 1860ttgtcgatat caaagctgaa ggaatttacc ggcagggaga agcctacagt cattacatga 1920gctgcatgga tggctaggta gcacgtatat tcttttattt tatgtgatgg cacgtccatt 1980ttgctaataa agtaataatg taaagaagtc attacgcaga gtactagtct tttattatgc 2040gatgcaacaa tcactcagtt ttgctattaa aaatgggact cacttctttc ccagaaaaaa 2100aaaaaaaaaa aa 21122639PRTTypha latifolia 2Ala Pro Ala Arg Tyr Arg His Leu Ser Leu Ser Gln Ser Leu Ser Pro1 5 10 15Tyr Leu Gln Glu Met Ala Met Ser Val Pro Thr Met Arg Leu His Pro 20 25 30Leu Val Pro Ser Ser Lys Leu Leu Ser Pro Ser Ser Ser Ser Pro Ala 35 40 45Val Leu Val Ser Ser Arg Ile Pro Leu Leu Ser Leu Arg Arg Pro Asn 50 55 60Leu Arg Phe Ser Val Lys Ala Thr Ala Ser Ser Thr Pro Ser Thr Ala65 70 75 80Glu Ser Ile Lys Ile Lys Ser Ile Pro Thr Lys Pro Val Glu Gly Gln 85 90 95Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val Phe Gln Gln 100 105 110Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn Ser Leu Pro 115 120 125Leu Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly Asp Gly Arg 130 135 140Tyr Phe Asn Arg Glu Ala Ala Gln Ile Ile Ile Lys Ile Ala Ala Gly145 150 155 160Asn Gly Val Gly Lys Ile Leu Val Gly Arg Asp Gly Ile Met Ser Thr 165 170 175Pro Ala Val Ser Ala Val Ile Arg Lys Gln Lys Ala Asn Gly Gly Phe 180 185 190Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Asp Tyr Asp Trp Gly 195 200 205Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu Ser Ile Thr 210 215 220Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile Lys Ile Ser225 230 235 240Asp Ile Pro Asp Ile Asp Leu Ser Ser Leu Gly Val Thr Asn Tyr Gly 245 250 255Asn Phe Ser Val Glu Val Val Asp Pro Val Ser Asp Tyr Leu Glu Leu 260 265 270Met Glu Asn Val Phe Asp Phe Gln Leu Ile Lys Gly Leu Leu Ser Arg 275 280 285Ser Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val Thr Gly Ala 290 295 300Tyr Ala Lys Pro Ile Phe Val Glu Arg Leu Arg Ala Ser Pro Asp Cys305 310 315 320Val Leu Asn Gly Val Pro Leu Glu Asp Phe Gly His Gly His Pro Asp 325 330 335Pro Asn Leu Thr Tyr Ala Lys Glu Leu Val Asp Val Met Tyr Thr Thr 340 345 350Asp Ala Pro Asp Leu Gly Ala Ala Ser Asp Gly Asp Gly Asp Arg Asn 355 360 365Met Ile Leu Gly Arg Arg Phe Phe Val Thr Pro Ser Asp Ser Val Ala 370 375 380Met Ile Ala Ala Asn Ala Gln Ala Ala Ile Pro Tyr Phe Gln Ala Gly385 390 395 400Pro Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu Asp Arg 405 410 415Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro Thr Gly Trp 420 425 430Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Lys Leu Ser Ile Cys Gly 435 440 445Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys Asp Gly 450 455 460Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile Ala Tyr Arg Asn Lys465 470 475 480Asp Lys Lys Ile Gly Glu Lys Leu Val Ser Val Glu Asp Ile Ala Lys 485 490 495Glu His Trp Ala Lys Tyr Gly Arg Asn Phe Phe Ser Arg Tyr Asp Tyr 500 505 510Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met Met Gln His Leu Arg 515 520 525Asp Phe Ile Ser Thr Ser Lys Pro Gly Glu Gln Tyr Gly Asn Tyr Thr 530 535 540Leu Gln Phe Ser Asp Asp Phe Ser Tyr Thr Asp Pro Val Asp Gly Ser545 550 555 560Val Ala Ser Lys Gln Gly Leu Arg Phe Val Phe Thr Asp Gly Ser Arg 565 570 575Val Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala Thr Ile Arg 580 585 590Ile Tyr Val Glu Gln Phe Glu Pro Asp Val Ser Lys His Asp Val Asp 595 600 605Ala Gln Ala Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu Ser Ile Ser 610 615 620Lys Leu Lys Glu Phe Thr Gly Arg Glu Lys Pro Thr Val Ile Thr625 630 63531951DNAZea mays 3ccacgcgtcc gcacaaactg ccctcgcggc ctcgcccgtc gcccctctcg atcacttctc 60tcccgacact ctctcactcc cgtgtcgtgt ctagcgccga cggcgttgct accggagccg 120gccagcggcc acgatgccta caatgcacgc gcttcgccta tgcccgctgc tctccaccat 180ccgatccaca ccaccgcggg ccactgccgc agcccgccag ggcgcgctct tcgtcgcccg 240ctgctcctcc gccgggacgc cgtcagccgc ccaggcgctc aagatcagtt caatcccgac 300caagccagtt gaggggcaga agactgggac tagtggcctg aggaaaaagg tgaaagtatt 360ccagcaggag aactaccttg ctaattggat tcaggctcta ttcaattcct tgccccctga 420agattatgtg ggtgcaaccc ttgtacttgg gggtgatggc cggtacttta acaaggaggc 480tgctcagatc atcattaaga ttgcagctgg aaatggagtt cagaagatca tagttggcag 540gaatggtcta ctgtcaacac ctgctgtatc tgctgtaatt cgtaaaagaa aagccaatgg 600cggctttatc atgagtgcaa gccataatcc aggtggacca gacaatgact ggggtattaa 660gtttaactac agcagtggac agccagcacc ggagacgatt actgatcaaa tttatggaaa 720cacactatca atttctgaaa taaaaacagc agacattcct gatactgatt tgtcctctgt 780tggagttgta agctatggtg atttcgccat agaagtgata gatcctgttt cagattacct 840tgaactaatg gagaatgtgt ttgacttcca acttatcaag gatttgcttt ctcggcctga 900tttcaggttc atatttgatg caatgcatgc aattactggt gcgtatgccg gacccatttt 960tgttgagaaa cttggagctg atccggactg catattaaat ggggtgcctc ttgaagattt 1020tggaaatggc catccagatc caaatctaac ttacgctaag gagcttgttt ttactatgtt 1080tggaacccat gcacctgact ttggtgcagc aagtgatggt gatggtgatc ggaacatgat 1140tcttgggaaa aggttcttta ttaccccatc agactctgtt gcaataattg cagccaatgc 1200acagacagca attccttatt tccagtttgg tacaaaagga ctcgcgagat caatgccaac 1260cagtggtgct cttgatcgtg ttgccgagaa attgaatgtt ccattctttg aggttccaac 1320aggctggaaa ttttttggca acctaatgga tgcaggaaaa ttgtctattt gtggagagga 1380aagttttggg actggatctg atcacatcag agagaaggat ggcatctggg ctgttctggc 1440ttggctttcc atacttgcac accggaacaa ggataagaag gtcggagaga gattagtgtc 1500agttgaagat attgctatgg agcactggaa aacctatgga aggaatttct tttctagata 1560cgattatgag gcgtgtgaat cacacagtgc aaaccagatg atggatcacc ttagagatgt 1620tatggcaaat agcaagcctg gagagaaata cggaaattac accctccaat ttgctgatga 1680tttcagctat actgatcctg tagacggtag tacggtatca aaacaaggac ttcgatttgt 1740tttcactgat ggatctagga ttatcttccg gctttcggga accggatctg ctggagctac 1800tatccgcctc tacatagaac aatttgaatc tgatatctcg aagcatagtc tcgatgctca 1860aacagctttg aagcctttaa tagacctggc tttgtctgtt tcgaagctca aggacttcac 1920aggaagagag aaacctactg tcataacata g 19514605PRTZea mays 4Met Pro Thr Met His Ala Leu Arg Leu Cys Pro Leu Leu Ser Thr Ile1 5 10 15Arg Ser Thr Pro Pro Arg Ala Thr Ala Ala Ala Arg Gln Gly Ala Leu 20 25 30Phe Val Ala Arg Cys Ser Ser Ala Gly Thr Pro Ser Ala Ala Gln Ala 35 40 45Leu Lys Ile Ser Ser Ile Pro Thr Lys Pro Val Glu Gly Gln Lys Thr 50 55 60Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val Phe Gln Gln Glu Asn65 70 75 80Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn Ser Leu Pro Pro Glu 85 90 95Asp Tyr Val Gly Ala Thr Leu Val Leu Gly Gly Asp Gly Arg Tyr Phe 100 105 110Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile Ala Ala Gly Asn Gly 115 120 125Val Gln Lys Ile Ile Val Gly Arg Asn Gly Leu Leu Ser Thr Pro Ala 130 135 140Val Ser Ala Val Ile Arg Lys Arg Lys Ala Asn Gly Gly Phe Ile Met145 150 155 160Ser Ala Ser His Asn Pro Gly Gly Pro Asp Asn Asp Trp Gly Ile Lys 165 170 175Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu Thr Ile Thr Asp Gln 180 185 190Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile Lys Thr Ala Asp Ile 195 200 205Pro Asp Thr Asp Leu Ser Ser Val Gly Val Val Ser Tyr Gly Asp Phe 210 215 220Ala Ile Glu Val Ile Asp Pro Val Ser Asp Tyr Leu Glu Leu Met Glu225 230 235 240Asn Val Phe Asp Phe Gln Leu Ile Lys Asp Leu Leu Ser Arg Pro Asp 245 250 255Phe Arg Phe Ile Phe Asp Ala Met His Ala Ile Thr Gly Ala Tyr Ala 260 265 270Gly Pro Ile Phe Val Glu Lys Leu Gly Ala Asp Pro Asp Cys Ile Leu 275 280 285Asn Gly Val Pro Leu Glu Asp Phe Gly Asn Gly His Pro Asp Pro Asn 290 295 300Leu Thr Tyr Ala Lys Glu Leu Val Phe Thr Met Phe Gly Thr His Ala305 310 315 320Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp Arg Asn Met Ile 325 330 335Leu Gly Lys Arg Phe Phe Ile Thr Pro Ser Asp Ser Val Ala Ile Ile 340 345 350Ala Ala Asn Ala Gln Thr Ala Ile Pro Tyr Phe Gln Phe Gly Thr Lys 355 360 365Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu Asp Arg Val Ala 370 375 380Glu Lys Leu Asn Val Pro Phe Phe Glu Val Pro Thr Gly Trp Lys Phe385 390 395 400Phe Gly Asn Leu Met Asp Ala Gly Lys Leu Ser Ile Cys Gly Glu Glu 405 410 415Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys Asp Gly Ile Trp 420 425 430Ala Val Leu Ala Trp Leu Ser Ile Leu Ala His Arg Asn Lys Asp Lys 435 440 445Lys Val Gly Glu Arg Leu Val Ser Val Glu Asp Ile Ala Met Glu His 450 455 460Trp Lys Thr Tyr Gly Arg Asn Phe Phe Ser Arg Tyr Asp Tyr Glu Ala465 470 475 480Cys Glu Ser His Ser Ala Asn Gln Met Met Asp His Leu Arg Asp Val 485 490 495Met Ala Asn Ser Lys Pro Gly Glu Lys Tyr Gly Asn Tyr Thr Leu Gln 500 505 510Phe Ala Asp Asp Phe Ser Tyr Thr Asp Pro Val Asp Gly Ser Thr Val 515 520 525Ser Lys Gln Gly Leu Arg Phe Val Phe Thr Asp Gly Ser Arg Ile Ile 530 535 540Phe Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala Thr Ile Arg Leu Tyr545 550 555 560Ile Glu Gln Phe Glu Ser Asp Ile Ser Lys His Ser Leu Asp Ala Gln 565 570 575Thr Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu Ser Val Ser Lys Leu 580 585 590Lys Asp Phe Thr Gly Arg Glu Lys Pro Thr Val Ile Thr 595 600 6055573DNAOryza sativa 5tgatggagca tcttagagat gtgatcgcaa aaagcaagcc tggagagaaa tatggaaact 60atacccttca gtttgccgat gatttcagtt acactgatcc ggtggatggt agcactgtat 120ctaaacaagg gcttcgattt gtattcaccg atggatctag gattatcttc cgcctttcgg 180gaaccggatc tgctggagca acaatccgta tatacattga gcaattcgag tctgatgcct 240caaagcatga tctggatgca caaatagctt tgaagccttt aatagaccta gctctatctg 300tttcaaagtt gaaggacttc actgggaaga gataagccta ctgtcataac ataaacatac 360cggtgacatt agcaatgtta ccacctgggt attcttttat ttccttgttt ttaaaagccc 420cttccaaccg atgaaccaat aatgttatcc taagccaagt tttgtactga gttgatggca 480aactgtatcc tggggggtac tttcaattga acataagtat gcaaggaatg aataaagctt 540ttaaaagcaa aaaaaaaaaa aaaaaaaaaa aaa 5736117PRTOryza sativaUNSURE(108)Xaa can be any naturally occurring amino acid 6Met Glu His Leu Arg Asp Val Ile Ala Lys Ser Lys Pro Gly Glu Lys1 5 10 15Tyr Gly Asn Tyr Thr Leu Gln Phe Ala Asp Asp Phe Ser Tyr Thr Asp 20 25 30Pro Val Asp Gly Ser Thr Val Ser Lys Gln Gly Leu Arg Phe Val Phe 35 40 45Thr Asp Gly Ser Arg Ile Ile Phe Arg Leu Ser Gly Thr Gly Ser Ala 50 55 60Gly Ala Thr Ile Arg Ile Tyr Ile Glu Gln Phe Glu Ser Asp Ala Ser65 70 75 80Lys His Asp Leu Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu 85 90 95Ala Leu Ser Val Ser Lys Leu Lys Asp Phe Thr Xaa Gly Arg Asp Lys 100 105 110Pro Thr Val Ile Thr 11572204DNAGlycine maxunsure(7)n = A, C, G, or T 7aaaactnttt ggaaccctcc agcatttcat ttctcatcat caatggcttt ctcttgtaaa 60cttgacagct tcattctctc tgcctataaa ccccaaaact ccattctccc actttcaatc 120caaccttcct ccttccttcc atctccttct tctttgaagc ctcagaagct tcccttcaga 180attcgctatg gttctaccat cagagccacg tcatcatcct caaccccttc cgcaaccatt 240gccgaacctg aaggcattaa gattaaatcg attccaacca agcccattga tggacaaaag 300actggaacca gtgggcttcg aaagaaggtg aaagtgttta tgcaagacaa ttaccttgca 360aattggatcc aggctctgtt taattcattg ccaccggagg actacaagaa tggtttgttg 420gtgttgggag gtgatggtcg atactttaat caggaagctg cacagataat aatcaaaatt 480gctgctggaa atggtgttgg aaaaattctg gttggaaagg aaggtatttt gtcaacacca 540gccgtttctg ctgttataag aaagagaaag gcaaatggtg gatttattat gagtgcaagc 600cataatcctg gcggacctga atatgattgg ggtattaagt ttaattacag cagtggacaa 660cctgcaccag aatccatcac tgacaagatt tatggaaata ccctgtcgat ctctgagata 720aagatagctg acattcctga tgttgattta tcaaaagttg gggttacaaa ttttggaagc 780ttcagtgtgg aagtaataga cccagtttct gactatctgg agctattgga gacagtattt 840gattttcagc taatcagagg tcttctttca cgtccagatt ttaggtttat atttgatgcc 900atgcatgcag ttactggtgc ttatgctaaa cccatcttcg ttgataaact cggtgctagt 960ctggattcaa tttcaaatgg aatccctttg gaagattttg gacatggcca tcctgatcct 1020aatctaacat atgcgaagga tcttgtcgac attctgtatg ctgaaaatgg acctgatttt 1080ggagctgcca gtgatgggga tggtgataga aatatgattt taggaagaag tttctttgta 1140actccttcag actctgtagc agttattgca gccaatgcaa gagaagcgat tccatacttc 1200aagaacggtg ttaagggtct tgctcgatca atgccaccaa gcggtgctct ggaccgtgtt 1260gctaaaaaat tgaacctccc tttctttgag gtccccactg gttggaaatt ttttgggaat 1320cttatggatg cnggaaattt gtccgtttgc ggggaagaga gttttggaac aggttctgat 1380cacattcgtg agaaagatgg catctgggct gtcttagctt ggctttctat tattgcacat 1440cgcaacaaag acaagaatcc cggggagaaa ttgatctccg tatctgacgt tgtgatggag 1500cactgggcaa cttatggaag gaatttcttc tctagatatg actacgagga atgtgaatct 1560gaaggtgcca ataagatgat agaataccta cgagatattt tgtctaagag caagcctggt 1620gatcagtatg gaagttatgt tctccagttt gcagatgatt ttacatacac cgatcctgta 1680gatggaagtg tggtatcaaa acaaggtgtt cggtttgttt ttacagacgg ttcaaggatt 1740atatatcgtt tatcaggaac tggttctgca ggggctacgg ttagagtgta cattgaacag 1800tttgaaccag atgtctctaa acatgatgtt gatgctcaaa ttgccttaaa accattaata 1860gatttggcaa

tatccgtgtc aaagctcaaa gacttcacag ggagggagaa gcctacagtc 1920atcacataat ggacaattcc acaaccactt gatcaagttg ttatatgttc caaggtgtgc 1980tctaagttga gtgcatacgc aggttgttta ttgcatgcct atccatatct gagctcgctc 2040gagttcggtc acttttggtt gttcaagaat tttggagcga taggtcccct gtaaaatatg 2100ctacttatat atttatgtgc aaagtatgaa gcaccgacgt gcaacaaaat aataataaaa 2160aagaatagtt tgctgctcta aggagctagg cctttcaaaa aaaa 22048628PRTGlycine max 8Met Ala Phe Ser Cys Lys Leu Asp Ser Phe Ile Leu Ser Ala Tyr Lys1 5 10 15Pro Gln Asn Ser Ile Leu Pro Leu Ser Ile Gln Pro Ser Ser Phe Leu 20 25 30Pro Ser Pro Ser Ser Leu Lys Pro Gln Lys Leu Pro Phe Arg Ile Arg 35 40 45Tyr Gly Ser Thr Ile Arg Ala Thr Ser Ser Ser Ser Thr Pro Ser Ala 50 55 60Thr Ile Ala Glu Pro Glu Gly Ile Lys Ile Lys Ser Ile Pro Thr Lys65 70 75 80Pro Ile Asp Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val 85 90 95Lys Val Phe Met Gln Asp Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu 100 105 110Phe Asn Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu 115 120 125Gly Gly Asp Gly Arg Tyr Phe Asn Gln Glu Ala Ala Gln Ile Ile Ile 130 135 140Lys Ile Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu145 150 155 160Gly Ile Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Lys 165 170 175Ala Asn Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro 180 185 190Glu Tyr Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala 195 200 205Pro Glu Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser 210 215 220Glu Ile Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Lys Val Gly225 230 235 240Val Thr Asn Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser 245 250 255Asp Tyr Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Arg 260 265 270Gly Leu Leu Ser Arg Pro Asp Phe Arg Phe Ile Phe Asp Ala Met His 275 280 285Ala Val Thr Gly Ala Tyr Ala Lys Pro Ile Phe Val Asp Lys Leu Gly 290 295 300Ala Ser Leu Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly305 310 315 320His Gly His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asp 325 330 335Ile Leu Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly 340 345 350Asp Gly Asp Arg Asn Met Ile Leu Gly Arg Ser Phe Phe Val Thr Pro 355 360 365Ser Asp Ser Val Ala Val Ile Ala Ala Asn Ala Arg Glu Ala Ile Pro 370 375 380Tyr Phe Lys Asn Gly Val Lys Gly Leu Ala Arg Ser Met Pro Pro Ser385 390 395 400Gly Ala Leu Asp Arg Val Ala Lys Lys Leu Asn Leu Pro Phe Phe Glu 405 410 415Val Pro Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn 420 425 430Leu Ser Val Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile 435 440 445Arg Glu Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile 450 455 460Ala His Arg Asn Lys Asp Lys Asn Pro Gly Glu Lys Leu Ile Ser Val465 470 475 480Ser Asp Val Val Met Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe 485 490 495Ser Arg Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met 500 505 510Ile Glu Tyr Leu Arg Asp Ile Leu Ser Lys Ser Lys Pro Gly Asp Gln 515 520 525Tyr Gly Ser Tyr Val Leu Gln Phe Ala Asp Asp Phe Thr Tyr Thr Asp 530 535 540Pro Val Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe Val Phe545 550 555 560Thr Asp Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala 565 570 575Gly Ala Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp Val Ser 580 585 590Lys His Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu 595 600 605Ala Ile Ser Val Ser Lys Leu Lys Asp Phe Thr Gly Arg Glu Lys Pro 610 615 620Thr Val Ile Thr62592197DNAOryza sativa 9gcacgaggct tgcccgcttc cttccgcggt gcaagcgcaa caccacctca cctcactccc 60cttctcgcct cttctcccct tctccacctc ctcttctctc cgcgtggcgg tggcattgcc 120ggccgccgca tcgtctcggg atggcctcgc acgcgctccg cctccacccg ctgctcttct 180ccgccgccgc cgcgcgcccg gctccgctcg cggcgcggcc cggtggtggt gcccgccggg 240tccaccgccg ccactctctc gccgtcgtcc ggtgctcctc ctccgccgcc caggcgctca 300agatcaagtc gattccgacc aagcccgttg aggggcagaa gaccgggacc agtgggttga 360ggaagaaggt gaaagtgttc cagcaggaga attacctcgc taattggatt caggctctgt 420tcaattcatt gcccccggag gattatgttg gtggaaccct tgtgcttggt ggtgatggcc 480gatactttaa caaggatgct gctcagatta tcactaaaat tgcagctggg aatggtgttg 540ggaagatcct agttggcagg aacggtctgc tgtcaacgcc tgctgtatct gcagtaattc 600gtaaaagaca agccaatggt ggcttcatca tgagtgcaag ccataatcca ggtgggccag 660ataatgattg gggtatcaag ttcaactata gcagtgggca gccagcacca gagacaatta 720ccgaccaaat atatggaaac acactttcga tttctgaaat aaaaacggca gatattcctg 780atgttgattt gtcctctcta ggagttgtaa gctatggtga tttcaccgtt gaagtgatag 840accctgtctt ggactacctt gagctaatgg agaatgtgtt tgacttccaa cttatcaagg 900gcttgttgtc tcggccagat ttcaggtttg tatttgatgc catgcatgct gtgactggtg 960catatgcgga tcctattttt gttgagaaac ttggagctga tccggactat atattaaatg 1020gtgttccact tgaagatttt ggcaatggtc accctgatcc taatttaact tatgccaaag 1080agcttgtgtt taccatgttt ggaagcggag cacctgactt tggtgcagca agtgatggtg 1140atggtgatcg aaacatgatt cttggaagaa ggttctttgt tacaccatca gactctgttg 1200caataattgc agcgaatgca caggcagcaa ttccttattt ccaatctggt ccaaaaggtc 1260ttgctagatc aatgccaacg agtggtgctc ttgatcgtgt agctgataaa ttgaatgttc 1320cgttctttga ggtaccaaca ggatggaaat tttttggaaa cctaatggat gcaggtaaat 1380tgtctatatg tggagaggaa agttttggga caggatctga tcacatcagg gagaaggatg 1440gcatatgggc tgttctagct tggctgtcca tacttgcaca ccggaacaag gataagaagg 1500ccggggagag attagtgtca gtggaagatg tagctaggga acactgggca acctatggaa 1560ggaatttctt ctccagatat gattatgagg agtgtgaatc tgagagtgca aataagatga 1620tggagcatct tagagatgtg atcgcaaaaa gcaagcctgg agagaaatat ggaaactata 1680cccttcagtt tgccgatgat ttcagttaca ctgatccggt ggatggtagc actgtatcta 1740aacaagggct tcgatttgta ttcaccgatg gatctaggat tatcttccgc ctttcgggaa 1800ccggatctgc tggagcaaca atccgtatat acattgagca attcgagtct gatgcctcaa 1860agcatgatct ggatgcacaa atagctttga agcctttaat agacctagct ctatctgttt 1920caaagttgaa ggacttcact ggaagagata agcctactgt cataacataa acataccggt 1980gacattagca atgttaccac ctgtgtattc ttttatttct ttgtttttat agccccttcc 2040aaccgatgaa ccaataatgt aatcttaggc caagttttgt actgagttga tggcaaactg 2100tatcttggag gtacctttca ttgaacatag tatgcaggaa tgaataagct tttagagcaa 2160tggtacatat ttcagaacaa aaaaaaaaaa aaaaaaa 219710655PRTOryza sativa 10Thr Arg Leu Ala Arg Phe Leu Pro Arg Cys Lys Arg Asn Thr Thr Ser1 5 10 15Pro His Ser Pro Ser Arg Leu Phe Ser Pro Ser Pro Pro Pro Leu Leu 20 25 30Ser Ala Trp Arg Trp His Cys Arg Pro Pro His Arg Leu Gly Met Ala 35 40 45Ser His Ala Leu Arg Leu His Pro Leu Leu Phe Ser Ala Ala Ala Ala 50 55 60Arg Pro Ala Pro Leu Ala Ala Arg Pro Gly Gly Gly Ala Arg Arg Val65 70 75 80His Arg Arg His Ser Leu Ala Val Val Arg Cys Ser Ser Ser Ala Ala 85 90 95Gln Ala Leu Lys Ile Lys Ser Ile Pro Thr Lys Pro Val Glu Gly Gln 100 105 110Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val Phe Gln Gln 115 120 125Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn Ser Leu Pro 130 135 140Pro Glu Asp Tyr Val Gly Gly Thr Leu Val Leu Gly Gly Asp Gly Arg145 150 155 160Tyr Phe Asn Lys Asp Ala Ala Gln Ile Ile Thr Lys Ile Ala Ala Gly 165 170 175Asn Gly Val Gly Lys Ile Leu Val Gly Arg Asn Gly Leu Leu Ser Thr 180 185 190Pro Ala Val Ser Ala Val Ile Arg Lys Arg Gln Ala Asn Gly Gly Phe 195 200 205Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Asp Asn Asp Trp Gly 210 215 220Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu Thr Ile Thr225 230 235 240Asp Gln Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile Lys Thr Ala 245 250 255Asp Ile Pro Asp Val Asp Leu Ser Ser Leu Gly Val Val Ser Tyr Gly 260 265 270Asp Phe Thr Val Glu Val Ile Asp Pro Val Leu Asp Tyr Leu Glu Leu 275 280 285Met Glu Asn Val Phe Asp Phe Gln Leu Ile Lys Gly Leu Leu Ser Arg 290 295 300Pro Asp Phe Arg Phe Val Phe Asp Ala Met His Ala Val Thr Gly Ala305 310 315 320Tyr Ala Asp Pro Ile Phe Val Glu Lys Leu Gly Ala Asp Pro Asp Tyr 325 330 335Ile Leu Asn Gly Val Pro Leu Glu Asp Phe Gly Asn Gly His Pro Asp 340 345 350Pro Asn Leu Thr Tyr Ala Lys Glu Leu Val Phe Thr Met Phe Gly Ser 355 360 365Gly Ala Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp Arg Asn 370 375 380Met Ile Leu Gly Arg Arg Phe Phe Val Thr Pro Ser Asp Ser Val Ala385 390 395 400Ile Ile Ala Ala Asn Ala Gln Ala Ala Ile Pro Tyr Phe Gln Ser Gly 405 410 415Pro Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala Leu Asp Arg 420 425 430Val Ala Asp Lys Leu Asn Val Pro Phe Phe Glu Val Pro Thr Gly Trp 435 440 445Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Lys Leu Ser Ile Cys Gly 450 455 460Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu Lys Asp Gly465 470 475 480Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Leu Ala His Arg Asn Lys 485 490 495Asp Lys Lys Ala Gly Glu Arg Leu Val Ser Val Glu Asp Val Ala Arg 500 505 510Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe Ser Arg Tyr Asp Tyr 515 520 525Glu Glu Cys Glu Ser Glu Ser Ala Asn Lys Met Met Glu His Leu Arg 530 535 540Asp Val Ile Ala Lys Ser Lys Pro Gly Glu Lys Tyr Gly Asn Tyr Thr545 550 555 560Leu Gln Phe Ala Asp Asp Phe Ser Tyr Thr Asp Pro Val Asp Gly Ser 565 570 575Thr Val Ser Lys Gln Gly Leu Arg Phe Val Phe Thr Asp Gly Ser Arg 580 585 590Ile Ile Phe Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala Thr Ile Arg 595 600 605Ile Tyr Ile Glu Gln Phe Glu Ser Asp Ala Ser Lys His Asp Leu Asp 610 615 620Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu Ser Val Ser625 630 635 640Lys Leu Lys Asp Phe Thr Gly Arg Asp Lys Pro Thr Val Ile Thr 645 650 65511629PRTBrassica napus 11Met Ser Ser Thr Tyr Ala Arg Phe Asp Thr Val Phe Leu Leu Ser Arg1 5 10 15Phe Ala Gly Ala Lys Tyr Ser Pro Leu Trp Pro Ser Ser Ser Ser Ser 20 25 30Ser His Ser Ser Leu Leu Ser Ser Gly Ile His Leu Arg Ala Lys Pro 35 40 45Asn Ser Arg Leu Arg Ser Val Thr Gly Ala Ser Ser Ser Ser Ser Gly 50 55 60Pro Ile Ile Ala Gly Ser Glu Ser Ile Glu Ile Lys Ser Leu Pro Thr65 70 75 80Lys Pro Ile Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys 85 90 95Val Lys Val Phe Met Gln Asp Asn Tyr Leu Ala Asn Trp Ile Gln Ala 100 105 110Leu Phe Asn Ser Leu Pro Leu Glu Asp Tyr Lys Asp Ala Thr Leu Val 115 120 125Leu Gly Gly Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ser Gln Ile Ile 130 135 140Ile Lys Ile Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Gln145 150 155 160Glu Gly Ile Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg 165 170 175Lys Ala Asn Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly 180 185 190Pro Glu Tyr Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro 195 200 205Ala Pro Glu Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile 210 215 220Ser Glu Ile Lys Val Ala Glu Ile Pro Asp Ile Asp Leu Ser His Val225 230 235 240Gly Val Thr Lys Tyr Gly Asn Phe Ser Val Glu Val Ile Asp Pro Ile 245 250 255Ser Asp Tyr Leu Glu Leu Met Glu Asp Val Phe Asp Phe Asp Leu Ile 260 265 270Arg Gly Leu Leu Ser Arg Ser Asp Phe Gly Phe Met Phe Asp Ala Met 275 280 285His Ala Val Thr Gly Ala Tyr Ala Lys Pro Ile Phe Val Asp Asn Leu 290 295 300Glu Ala Lys Pro Asp Ser Ile Ser Asn Gly Val Pro Leu Glu Asp Phe305 310 315 320Gly His Gly His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val 325 330 335Asp Val Met Tyr Arg Asp Asp Gly Pro Asp Phe Gly Ala Ala Ser Asp 340 345 350Gly Asp Gly Asp Arg Asn Met Val Leu Gly Asn Lys Phe Phe Val Thr 355 360 365Pro Ser Asp Ser Val Ala Ile Ile Ala Ala Asn Ala Gln Glu Ala Ile 370 375 380Pro Tyr Phe Arg Ala Gly Pro Lys Gly Leu Ala Arg Ser Met Pro Thr385 390 395 400Ser Gly Ala Leu Asp Arg Val Ala Glu Lys Leu Lys Leu Pro Phe Phe 405 410 415Glu Val Pro Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly 420 425 430Lys Leu Ser Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His 435 440 445Ile Arg Glu Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile 450 455 460Leu Ala His Arg Ile Lys Asp Lys Lys Pro Gly Glu Lys Leu Val Ser465 470 475 480Val Ala Asp Val Val Asn Glu Tyr Trp Ala Thr Tyr Gly Arg Asn Phe 485 490 495Phe Ser Arg Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys 500 505 510Met Ile Glu Tyr Leu Arg Asp Ile Val Ala Lys Ser Lys Ala Gly Glu 515 520 525Asn Tyr Gly Asn Tyr Val Leu Gln Phe Ala Asp Asp Phe Ser Tyr Lys 530 535 540Asp Pro Val Asp Gly Ser Val Ala Ser Lys Gln Gly Val Arg Phe Val545 550 555 560Phe Thr Asp Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Asn Gly Ser 565 570 575Ala Gly Ala Thr Val Arg Ile Tyr Ile Glu Gln Phe Glu Pro Asp Val 580 585 590Ser Lys His Asp Val Asp Ala Gln Ile Ala Ile Lys Pro Leu Ile Asp 595 600 605Leu Ala Leu Ser Val Ser Lys Leu Lys Glu Phe Thr Gly Arg Glu Lys 610 615 620Pro Thr Val Ile Thr62512626PRTPisum sativum 12Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys1 5 10 15Pro Lys His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn 20 25 30Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn 35 40 45Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile 50 55 60Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile65 70 75 80Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val 85 90 95Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn 100 105 110Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly 115 120 125Asp Gly

Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile 130 135 140Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile145 150 155 160Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn 165 170 175Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr 180 185 190Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu 195 200 205Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile 210 215 220Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr225 230 235 240Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr 245 250 255Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu 260 265 270Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val 275 280 285Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Ser Ala Ser 290 295 300Leu Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly305 310 315 320His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Lys Ile Met 325 330 335Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly 340 345 350Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp 355 360 365Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe 370 375 380Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala385 390 395 400Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro 405 410 415Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser 420 425 430Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu 435 440 445Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile Ala His 450 455 460Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu Val Ser Val Ser Asp465 470 475 480Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe Ser Arg 485 490 495Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met Ile Glu 500 505 510Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro Gly Asp Lys Tyr Gly 515 520 525Ser Tyr Val Leu Gln Phe Ala Asp Asp Phe Thr Tyr Thr Asp Pro Val 530 535 540Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe Val Phe Thr Asp545 550 555 560Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala 565 570 575Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp Val Ser Lys His 580 585 590Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu 595 600 605Ser Val Ser Lys Leu Lys Asp Phe Thr Gly Arg Glu Lys Pro Thr Val 610 615 620Ile Thr62513626PRTPisum sativum 13Met Ala Phe Cys Tyr Arg Leu Asp Asn Phe Ile Ile Ser Ala Phe Lys1 5 10 15Pro Lys His Ser Asn Val Pro Leu Ser Ile His His Ser Ser Ser Asn 20 25 30Phe Pro Ser Phe Lys Val Gln Asn Phe Pro Phe Arg Val Arg Tyr Asn 35 40 45Ser Ala Ile Arg Ala Thr Ser Ser Ser Ser Ser Thr Pro Thr Thr Ile 50 55 60Ala Glu Pro Asn Asp Ile Lys Ile Asn Ser Ile Pro Thr Lys Pro Ile65 70 75 80Glu Gly Gln Lys Thr Gly Thr Ser Gly Leu Arg Lys Lys Val Lys Val 85 90 95Phe Lys Gln Glu Asn Tyr Leu Ala Asn Trp Ile Gln Ala Leu Phe Asn 100 105 110Ser Leu Pro Pro Glu Asp Tyr Lys Asn Gly Leu Leu Val Leu Gly Gly 115 120 125Asp Gly Arg Tyr Phe Asn Lys Glu Ala Ala Gln Ile Ile Ile Lys Ile 130 135 140Ala Ala Gly Asn Gly Val Gly Lys Ile Leu Val Gly Lys Glu Gly Ile145 150 155 160Leu Ser Thr Pro Ala Val Ser Ala Val Ile Arg Lys Arg Glu Ala Asn 165 170 175Gly Gly Phe Ile Met Ser Ala Ser His Asn Pro Gly Gly Pro Glu Tyr 180 185 190Asp Trp Gly Ile Lys Phe Asn Tyr Ser Ser Gly Gln Pro Ala Pro Glu 195 200 205Ser Ile Thr Asp Lys Ile Tyr Gly Asn Thr Leu Ser Ile Ser Glu Ile 210 215 220Lys Ile Ala Asp Ile Pro Asp Val Asp Leu Ser Asn Val Gly Val Thr225 230 235 240Lys Phe Gly Ser Phe Ser Val Glu Val Ile Asp Pro Val Ser Asp Tyr 245 250 255Leu Glu Leu Leu Glu Thr Val Phe Asp Phe Gln Leu Ile Lys Ser Leu 260 265 270Ile Ser Arg Pro Asp Phe Arg Phe Thr Phe Asp Ala Met His Ala Val 275 280 285Ala Gly Ala Tyr Ala Thr Pro Ile Phe Val Asp Lys Leu Gly Ala Ser 290 295 300Pro Asp Ser Ile Ser Asn Gly Ile Pro Leu Glu Asp Phe Gly His Gly305 310 315 320His Pro Asp Pro Asn Leu Thr Tyr Ala Lys Asp Leu Val Asn Ile Met 325 330 335Tyr Ala Glu Asn Gly Pro Asp Phe Gly Ala Ala Ser Asp Gly Asp Gly 340 345 350Asp Arg Asn Met Ile Leu Gly Thr Ser Phe Phe Val Thr Pro Ser Asp 355 360 365Ser Val Ala Val Ile Ala Ala Asn Ala Lys Glu Ala Ile Pro Tyr Phe 370 375 380Lys Asp Ser Ile Lys Gly Leu Ala Arg Ser Met Pro Thr Ser Gly Ala385 390 395 400Leu Asp Arg Val Ala Glu Lys Leu Asn Leu Pro Phe Phe Glu Val Pro 405 410 415Thr Gly Trp Lys Phe Phe Gly Asn Leu Met Asp Ala Gly Asn Leu Ser 420 425 430Ile Cys Gly Glu Glu Ser Phe Gly Thr Gly Ser Asp His Ile Arg Glu 435 440 445Lys Asp Gly Ile Trp Ala Val Leu Ala Trp Leu Ser Ile Ile Ala His 450 455 460Arg Asn Lys Asp Thr Lys Pro Gly Glu Lys Leu Val Ser Val Ser Asp465 470 475 480Val Val Lys Glu His Trp Ala Thr Tyr Gly Arg Asn Phe Phe Ser Arg 485 490 495Tyr Asp Tyr Glu Glu Cys Glu Ser Glu Gly Ala Asn Lys Met Ile Glu 500 505 510Tyr Leu Arg Glu Leu Leu Ser Lys Ser Lys Pro Gly Asp Lys Tyr Gly 515 520 525Ser Tyr Val Leu Gln Phe Ala Asp Asp Tyr Thr Tyr Thr Asp Pro Val 530 535 540Asp Gly Ser Val Val Ser Lys Gln Gly Val Arg Phe Val Phe Thr Asp545 550 555 560Gly Ser Arg Ile Ile Tyr Arg Leu Ser Gly Thr Gly Ser Ala Gly Ala 565 570 575Thr Val Arg Val Tyr Ile Glu Gln Phe Glu Pro Asp Val Ser Lys His 580 585 590Asp Val Asp Ala Gln Ile Ala Leu Lys Pro Leu Ile Asp Leu Ala Leu 595 600 605Ser Val Ser Lys Leu Lys Asp Phe Thr Gly Arg Glu Lys Pro Thr Val 610 615 620Ile Thr62514574DNAArtificial Sequence541 nt Glycine max fragment with 5' and 3' linkers 14ggccgctgag ctgatttaag atttatcaaa agttggggtt acaaattttg gaagcttcag 60tgtggaagta atagacccag tttctgacta tctggagcta ttggagacag tatttgattt 120tcagctaatc agaggtcttc tttcacgtcc agattttagg tttatatttg atgccatgca 180tgcagttact ggtgcttatg ctaaacccat cttcgttgat aaactcggtg ctagtctgga 240ttcaatttca aatggaatcc ctttggaaga ttttggacat ggccatcctg atcctaatct 300aacatatgcg aaggatcttg tcgacattct gtatgctgaa aatggacctg attttggagc 360tgccagtgat ggggatggtg atagaaatat gattttagga agaagtttct ttgtaactcc 420ttcagactct gtagcagtta ttgcagccaa tgcaagagaa gcgattccat acttcaagaa 480cggtgttaag ggtcttgctc gatcaatgcc aacaagcggt gctctggacc gtgctgctaa 540aaaattgaac ctccctttct gagctgattt aagc 57415541DNAGlycine max 15gatttatcaa aagttggggt tacaaatttt ggaagcttca gtgtggaagt aatagaccca 60gtttctgact atctggagct attggagaca gtatttgatt ttcagctaat cagaggtctt 120ctttcacgtc cagattttag gtttatattt gatgccatgc atgcagttac tggtgcttat 180gctaaaccca tcttcgttga taaactcggt gctagtctgg attcaatttc aaatggaatc 240cctttggaag attttggaca tggccatcct gatcctaatc taacatatgc gaaggatctt 300gtcgacattc tgtatgctga aaatggacct gattttggag ctgccagtga tggggatggt 360gatagaaata tgattttagg aagaagtttc tttgtaactc cttcagactc tgtagcagtt 420attgcagcca atgcaagaga agcgattcca tacttcaaga acggtgttaa gggtcttgct 480cgatcaatgc caacaagcgg tgctctggac cgtgctgcta aaaaattgaa cctccctttc 540t 541162401DNAZea mays 16ccacgcgtcc gcacaaactg ccctcgcggc ctcgcccgtc gcccctctcg atcacttctc 60tcccgacact ctctcactcc cgtgtcgtgt ctagcgccga cggcgttgct accggagccg 120gccagcggcc acgatgccta caatgcacgc gcttcgccta tgcccgctgc tctccaccat 180ccgatccaca ccaccgcggg ccactgccgc agcccgccag ggcgcgctct tcgtcgcccg 240ctgctcctcc gccgggacgc cgtcagccgc ccaggcgctc aagatcagtt caatcccgac 300caagccagtt gaggggcaga agactgggac tagtggcctg aggaaaaagg tgaaagtatt 360ccagcaggag aactaccttg ctaattggat tcaggctcta ttcaattcct tgccccctga 420agattatgtg ggtgcaaccc ttgtacttgg gggtgatggc cggtacttta acaaggaggc 480tgctcagatc atcattaaga ttgcagctgg aaatggagtt cagaagatca tagttggcag 540gaatggtcta ctgtcaacac ctgctgtatc tgctgtaatt cgtaaaagaa aagccaatgg 600cggctttatc atgagtgcaa gccataatcc aggtggacca gacaatgact ggggtattaa 660gtttaactac agcagtggac agccagcacc ggagacgatt actgatcaaa tttatggaaa 720cacactatca atttctgaaa taaaaacagc agacattcct gatactgatt tgtcctctgt 780tggagttgta agctatggtg atttcgccat agaagtgata gatcctgttt cagattacct 840tgaactaatg gagaatgtgt ttgacttcca acttatcaag gatttgcttt ctcggcctga 900tttcaggttc atatttgatg caatgcatgc aattactggt gcgtatgccg gacccatttt 960tgttgagaaa cttggagctg atccggactg catattaaat ggggtgcctc ttgaagattt 1020tggaaatggc catccagatc caaatctaac ttacgctaag gagcttgttt ttactatgtt 1080tggaacccat gcacctgact ttggtgcagc aagtgatggt gatggtgatc ggaacatgat 1140tcttgggaaa aggttcttta ttaccccatc agactctgtt gcaataattg cagccaatgc 1200acagacagca attccttatt tccagtttgg tacaaaagga ctcgcgagat caatgccaac 1260cagtggtgct cttgatcgtg ttgccgagaa attgaatgtt ccattctttg aggttccaac 1320aggctggaaa ttttttggca acctaatgga tgcaggaaaa ttgtctattt gtggagagga 1380aagttttggg actggatctg atcacatcag agagaaggat ggcatctggg ctgttctggc 1440ttggctttcc atacttgcac accggaacaa ggataagaag gtcggagaga gattagtgtc 1500agttgaagat attgctatgg agcactggaa aacctatggc aggaatttct tttctagata 1560cgattatgag gcgtgtgaat cacacagtgc aaaccagatg atggatcacc ttagagatgt 1620tatggcaaat agcaagcctg gagagaaata cggaaattac accctccaat ttgctgatga 1680tttcagctat actgatcctg tagacggtag tacggtatca aaacaaggac ttcgatttgt 1740tttcactgat ggatctagga ttatcttccg gctttcggga accggatctg ctggagctac 1800tatccgcctc tacatagaac aatttgaatc tgatatctcg aagcatagtc tcgatgctca 1860aacagctttg aagcctttaa tagacctggc tttgtctgtt tcgaagctca aggacttcac 1920aggaagagag aaacctactg tcataacata ggccctgttt gtttcggctt ttggcagctt 1980ctggccacca aaagctactg cgtactgtca aacgctcagc ttttcagcca gcttctataa 2040aattcgttgg gggcaaaaac catctaaaat caaataaaca cataatcggt tgagtcgttg 2100taatagtagg aattcatcac tttctagatc ctgagcctta tgaacaactt tatcttccta 2160cacacataat cgtaatgata ctcagattct cccacagcca gattctcccc acagccagat 2220tttcagaaaa gttggtcaga aaaaagctga accaaacagc cccataatat ttagatgttg 2280ttgtcctcgg ccataccaac tgagcagcat gggccaagaa ttgaactgat ggaaaatatg 2340tatcattagg acaaattccg ccagaataag ttgttcctcg gaaaaaaaaa aaaaaaaaaa 2400g 2401174974DNAArtificial Sequenceplasmid pKS133 17ggccgccgac tcgacgatga gcgagatgac cagctccggc cgcgacacaa gtgtgagagt 60actaaataaa tgctttggtt gtacgaaatc attacactaa ataaaataat caaagcttat 120atatgccttc cgctaaggcc gaatgcaaag aaattggttc tttctcgtta tcttttgcca 180cttttactag tacgtattaa ttactactta atcatctttg tttacggctc attatatccg 240tcgacggcgc gcccgatcat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga 300cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact 360cagcttcctt tcgggctttg ttagcagccg gatcgatcca agctgtacct cactattcct 420ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc 480catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc 540cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt 600caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc cggagccgcg 660gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag 720ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg 780cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc 840cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct 900catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat 960acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc 1020cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat 1080ccatagcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc aggtcttgca 1140acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg aattccccaa 1200tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat 1260ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat 1320cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt 1380cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc ttttccatgg 1440gtatatctcc ttcttaaagt taaacaaaat tatttctaga gggaaaccgt tgtggtctcc 1500ctatagtgag tcgtattaat ttcgcgggat cgagatctga tcaacctgca ttaatgaatc 1560ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 1620gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 1680atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 1740caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 1800cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 1860taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 1920ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc 1980tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 2040gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 2100ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 2160aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 2220aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 2280agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 2340cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 2400gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgacatt aacctataaa 2460aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 2520tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 2580caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 2640gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc 2700tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctatagaacg 2760gcgcgccaag cttggatcct cgaagagaag ggttaataac acatttttta acatttttaa 2820cacaaatttt agttatttaa aaatttatta aaaaatttaa aataagaaga ggaactcttt 2880aaataaatct aacttacaaa atttatgatt tttaataagt tttcaccaat aaaaaatgtc 2940ataaaaatat gttaaaaagt atattatcaa tattctcttt atgataaata aaaagaaaaa 3000aaaaataaaa gttaagtgaa aatgagattg aagtgacttt aggtgtgtat aaatatatca 3060accccgccaa caatttattt aatccaaata tattgaagta tattattcca tagcctttat 3120ttatttatat atttattata taaaagcttt atttgttcta ggttgttcat gaaatatttt 3180tttggtttta tctccgttgt aagaaaatca tgtgctttgt gtcgccactc actattgcag 3240ctttttcatg cattggtcag attgacggtt gattgtattt ttgtttttta tggttttgtg 3300ttatgactta agtcttcatc tctttatctc ttcatcaggt ttgatggtta cctaatatgg 3360tccatgggta catgcatggt taaattaggt ggccaacttt gttgtgaacg atagaatttt 3420ttttatatta agtaaactat ttttatatta tgaaataata ataaaaaaaa tattttatca 3480ttattaacaa aatcatatta gttaatttgt taactctata ataaaagaaa tactgtaaca 3540ttcacattac atggtaacat ctttccaccc tttcatttgt tttttgtttg atgacttttt 3600ttcttgttta aatttatttc ccttctttta aatttggaat acattatcat catatataaa 3660ctaaaatact aaaaacagga ttacacaaat gataaataat aacacaaata tttataaatc 3720tagctgcaat atatttaaac tagctatatc gatattgtaa aataaaacta gctgcattga 3780tactgataaa aaaatatcat gtgctttctg gactgatgat gcagtatact tttgacattg 3840cctttatttt atttttcaga aaagctttct tagttctggg ttcttcatta tttgtttccc 3900atctccattg tgaattgaat catttgcttc gtgtcacaaa tacaatttag ntaggtacat 3960gcattggtca gattcacggt ttattatgtc atgacttaag ttcatggtag tacattacct 4020gccacgcatg cattatattg gttagatttg ataggcaaat ttggttgtca acaatataaa 4080tataaataat gtttttatat tacgaaataa cagtgatcaa aacaaacagt tttatcttta 4140ttaacaagat tttgtttttg tttgatgacg ttttttaatg tttacgcttt cccccttctt 4200ttgaatttag aacactttat catcataaaa tcaaatacta aaaaaattac atatttcata 4260aataataaca caaatatttt taaaaaatct gaaataataa tgaacaatat tacatattat 4320cacgaaaatt cattaataaa aatattatat aaataaaatg taatagtagt tatatgtagg 4380aaaaaagtac tgcacgcata atatatacaa aaagattaaa

atgaactatt ataaataata 4440acactaaatt aatggtgaat catatcaaaa taatgaaaaa gtaaataaaa tttgtaatta 4500acttctatat gtattacaca cacaaataat aaataatagt aaaaaaaatt atgataaata 4560tttaccatct cataagatat ttaaaataat gataaaaata tagattattt tttatgcaac 4620tagctagcca aaaagagaac acgggtatat ataaaaagag tacctttaaa ttctactgta 4680cttcctttat tcctgacgtt tttatatcaa gtggacatac gtgaagattt taattatcag 4740tctaaatatt tcattagcac ttaatacttt tctgttttat tcctatccta taagtagtcc 4800cgattctccc aacattgctt attcacacaa ctaactaaga aagtcttcca tagcccccca 4860agcggccgga gctggtcatc tcgctcatcg tcgagtcggc ggccggagct ggtcatctcg 4920ctcatcgtcg agtcggcggc cgccgactcg acgatgagcg agatgaccag ctcc 49741834DNAArtificial SequenceSynthetic DNA Linker 18ggcgcgccaa gcttggatcc gtcgacggcg cgcc 341980DNAArtificial SequenceSynthetic Complementary Region of pKS106 and pKS124 19cggccggagc tggtcatctc gctcatcgtc gagtcggcgg ccgccgactc gacgatgagc 60gagatgacca gctccggccg 8020154DNAArtificial SequenceSynthetic Complementary Region of pKS133 20cggccggagc tggtcatctc gctcatcgtc gagtcggcgg ccggagctgg tcatctcgct 60catcgtcgag tcggcggccg ccgactcgac gatgagcgag atgaccagct ccggccgccg 120actcgacgat gagcgagatg accagctccg gccg 1542192DNAArtificial SequenceSynthetic PCR primer 21gaattccggc cggagctggt catctcgctc atcgtcgagt cggcggccgc cgactcgacg 60atgagcgaga tgaccagctc cggccggaat tc 922215DNAArtificial SequenceSynthetic PCR primer 22gaattccggc cggag 15231874DNAGlycine maxCDS(42)..(1637) 23ccaaatccaa aaaggtttct atttccgtta ttgatttagc a atg gat tca gct tgt 56 Met Asp Ser Ala Cys 1 5 gca acc ctg aat ggc cgc cat cta gcc aaa gtt agt gag gga att gga 104Ala Thr Leu Asn Gly Arg His Leu Ala Lys Val Ser Glu Gly Ile Gly 10 15 20 aga aac aga aca agt ggc ttc tgg ggt gag agt acg agg gga agt gtg 152Arg Asn Arg Thr Ser Gly Phe Trp Gly Glu Ser Thr Arg Gly Ser Val 25 30 35 aac aca aaa agg ttt ttg agt gtt caa tca tgc aag act tca cga acc 200Asn Thr Lys Arg Phe Leu Ser Val Gln Ser Cys Lys Thr Ser Arg Thr 40 45 50 aat agg aat ctt aga aac tcc aag cct gga agt gga att gca cgc gct 248Asn Arg Asn Leu Arg Asn Ser Lys Pro Gly Ser Gly Ile Ala Arg Ala 55 60 65 gtt ctc aca tca gac atc gac gaa gat tcc atg gca ttt caa ggg gta 296Val Leu Thr Ser Asp Ile Asp Glu Asp Ser Met Ala Phe Gln Gly Val 70 75 80 85 ccc act ttt gag aaa cct gaa gtg gac cca aaa agt gtg gct tcc atc 344Pro Thr Phe Glu Lys Pro Glu Val Asp Pro Lys Ser Val Ala Ser Ile 90 95 100 ata ttg ggt gga ggt gca gga act cga ctc ttt cct ctt act ggc aga 392Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Phe Pro Leu Thr Gly Arg 105 110 115 aga gcc aag cca gcg gtt cca att gga ggg tgt tat aga ctc ata gat 440Arg Ala Lys Pro Ala Val Pro Ile Gly Gly Cys Tyr Arg Leu Ile Asp 120 125 130 atc ccc atg agc aat tgc atc aat agt ggc atc aga aaa att ttc atc 488Ile Pro Met Ser Asn Cys Ile Asn Ser Gly Ile Arg Lys Ile Phe Ile 135 140 145 ttg acg cag ttc aat tct ttc tct ctc aac cgc cac ctg tcc cgt gca 536Leu Thr Gln Phe Asn Ser Phe Ser Leu Asn Arg His Leu Ser Arg Ala 150 155 160 165 tac agc ttc gga aat ggc atg act ttt gga gat ggg ttt gtg gag gtc 584Tyr Ser Phe Gly Asn Gly Met Thr Phe Gly Asp Gly Phe Val Glu Val 170 175 180 ttg gca gct act caa aca ccg ggt gag gct ggg aag aag tgg ttc caa 632Leu Ala Ala Thr Gln Thr Pro Gly Glu Ala Gly Lys Lys Trp Phe Gln 185 190 195 ggg aca gct gat gct gta aga caa ttt ata tgg gtt ttt gag gat gcc 680Gly Thr Ala Asp Ala Val Arg Gln Phe Ile Trp Val Phe Glu Asp Ala 200 205 210 aag aac aag aat gtt gag cat ata ttg ata ctt tct ggc gat cat ctt 728Lys Asn Lys Asn Val Glu His Ile Leu Ile Leu Ser Gly Asp His Leu 215 220 225 tac cgt atg gac tat atg gac ttt gta cag aga cat gtt gac aca aat 776Tyr Arg Met Asp Tyr Met Asp Phe Val Gln Arg His Val Asp Thr Asn 230 235 240 245 gcc gat atc aca gtt tca tgt gta ccc atg gat gac agt cgg gca tca 824Ala Asp Ile Thr Val Ser Cys Val Pro Met Asp Asp Ser Arg Ala Ser 250 255 260 gac tat gga ctg atg aaa att gat aaa aca gga cgg att ata cag ttt 872Asp Tyr Gly Leu Met Lys Ile Asp Lys Thr Gly Arg Ile Ile Gln Phe 265 270 275 gca gaa aaa cct aag gga tca gat cta aag gca atg cgt gtt gac acc 920Ala Glu Lys Pro Lys Gly Ser Asp Leu Lys Ala Met Arg Val Asp Thr 280 285 290 act ctt tta ggg tta ttg cca caa gaa gca gaa aaa cat cct tat att 968Thr Leu Leu Gly Leu Leu Pro Gln Glu Ala Glu Lys His Pro Tyr Ile 295 300 305 gca tcc atg ggt gtc tac gtg ttt aga act gaa acc ttg ctg caa cta 1016Ala Ser Met Gly Val Tyr Val Phe Arg Thr Glu Thr Leu Leu Gln Leu 310 315 320 325 tta aga tgg aaa tgt tct tca tgc aat gac ttt gga tct gaa att atc 1064Leu Arg Trp Lys Cys Ser Ser Cys Asn Asp Phe Gly Ser Glu Ile Ile 330 335 340 cca tct gct gtg aat gag cac aat gtc cag gca tat ttg ttc aat gac 1112Pro Ser Ala Val Asn Glu His Asn Val Gln Ala Tyr Leu Phe Asn Asp 345 350 355 tac tgg gaa gat att gga act ata aag tcc ttc ttt gat gca aat ctt 1160Tyr Trp Glu Asp Ile Gly Thr Ile Lys Ser Phe Phe Asp Ala Asn Leu 360 365 370 gct cta aca gaa cag cct cct aaa ttt gaa ttc tat gat cca aag aca 1208Ala Leu Thr Glu Gln Pro Pro Lys Phe Glu Phe Tyr Asp Pro Lys Thr 375 380 385 cct ttc ttc act tcc ccc aga ttc cta cca cct acc aaa gta gaa aaa 1256Pro Phe Phe Thr Ser Pro Arg Phe Leu Pro Pro Thr Lys Val Glu Lys 390 395 400 405 tgc aag att gtg gat gca att ata tct cat ggt tgc ttc ttg agg gag 1304Cys Lys Ile Val Asp Ala Ile Ile Ser His Gly Cys Phe Leu Arg Glu 410 415 420 tgc agc gtt caa cat tct att gtt gga gta cgc tca cgt ttg gag tct 1352Cys Ser Val Gln His Ser Ile Val Gly Val Arg Ser Arg Leu Glu Ser 425 430 435 ggt gtg gag ctt cag gat acg atg atg atg ggt gct gac tat tat caa 1400Gly Val Glu Leu Gln Asp Thr Met Met Met Gly Ala Asp Tyr Tyr Gln 440 445 450 act gag tat gaa att gca tct ctg gtg gca gaa ggg aag gtt cca att 1448Thr Glu Tyr Glu Ile Ala Ser Leu Val Ala Glu Gly Lys Val Pro Ile 455 460 465 ggt gtc ggg gca aat act aaa atc agg aat tgc ata atc gac aag aat 1496Gly Val Gly Ala Asn Thr Lys Ile Arg Asn Cys Ile Ile Asp Lys Asn 470 475 480 485 gcc aag ata gga aga aat gtg atc ata gca aac acc gat ggt gtt caa 1544Ala Lys Ile Gly Arg Asn Val Ile Ile Ala Asn Thr Asp Gly Val Gln 490 495 500 gaa gct gac agg gca aag gaa gga ttc tac att agg tcg ggc atc aca 1592Glu Ala Asp Arg Ala Lys Glu Gly Phe Tyr Ile Arg Ser Gly Ile Thr 505 510 515 gtt aca tta aaa aat gca aca atc aaa gat gga aca gtt ata tga 1637Val Thr Leu Lys Asn Ala Thr Ile Lys Asp Gly Thr Val Ile 520 525 530 agcacttcaa gttatccagc aggccacttt ataatagttt tgacataagt acattcactt 1697ccgacataat agtagatgaa atgctagctc gtacattaca aagtttctct cgatatatct 1757ctataatagt tatgtttatt gcatttgtag gacacttcac ttgtaataac aggcaattct 1817tgccactacg attaagttat aaattaaagt ttctaactca aaaaaaaaaa aaaaaaa 187424531PRTGlycine max 24Met Asp Ser Ala Cys Ala Thr Leu Asn Gly Arg His Leu Ala Lys Val 1 5 10 15 Ser Glu Gly Ile Gly Arg Asn Arg Thr Ser Gly Phe Trp Gly Glu Ser 20 25 30 Thr Arg Gly Ser Val Asn Thr Lys Arg Phe Leu Ser Val Gln Ser Cys 35 40 45 Lys Thr Ser Arg Thr Asn Arg Asn Leu Arg Asn Ser Lys Pro Gly Ser 50 55 60 Gly Ile Ala Arg Ala Val Leu Thr Ser Asp Ile Asp Glu Asp Ser Met 65 70 75 80 Ala Phe Gln Gly Val Pro Thr Phe Glu Lys Pro Glu Val Asp Pro Lys 85 90 95 Ser Val Ala Ser Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Phe 100 105 110 Pro Leu Thr Gly Arg Arg Ala Lys Pro Ala Val Pro Ile Gly Gly Cys 115 120 125 Tyr Arg Leu Ile Asp Ile Pro Met Ser Asn Cys Ile Asn Ser Gly Ile 130 135 140 Arg Lys Ile Phe Ile Leu Thr Gln Phe Asn Ser Phe Ser Leu Asn Arg 145 150 155 160 His Leu Ser Arg Ala Tyr Ser Phe Gly Asn Gly Met Thr Phe Gly Asp 165 170 175 Gly Phe Val Glu Val Leu Ala Ala Thr Gln Thr Pro Gly Glu Ala Gly 180 185 190 Lys Lys Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Phe Ile Trp 195 200 205 Val Phe Glu Asp Ala Lys Asn Lys Asn Val Glu His Ile Leu Ile Leu 210 215 220 Ser Gly Asp His Leu Tyr Arg Met Asp Tyr Met Asp Phe Val Gln Arg 225 230 235 240 His Val Asp Thr Asn Ala Asp Ile Thr Val Ser Cys Val Pro Met Asp 245 250 255 Asp Ser Arg Ala Ser Asp Tyr Gly Leu Met Lys Ile Asp Lys Thr Gly 260 265 270 Arg Ile Ile Gln Phe Ala Glu Lys Pro Lys Gly Ser Asp Leu Lys Ala 275 280 285 Met Arg Val Asp Thr Thr Leu Leu Gly Leu Leu Pro Gln Glu Ala Glu 290 295 300 Lys His Pro Tyr Ile Ala Ser Met Gly Val Tyr Val Phe Arg Thr Glu 305 310 315 320 Thr Leu Leu Gln Leu Leu Arg Trp Lys Cys Ser Ser Cys Asn Asp Phe 325 330 335 Gly Ser Glu Ile Ile Pro Ser Ala Val Asn Glu His Asn Val Gln Ala 340 345 350 Tyr Leu Phe Asn Asp Tyr Trp Glu Asp Ile Gly Thr Ile Lys Ser Phe 355 360 365 Phe Asp Ala Asn Leu Ala Leu Thr Glu Gln Pro Pro Lys Phe Glu Phe 370 375 380 Tyr Asp Pro Lys Thr Pro Phe Phe Thr Ser Pro Arg Phe Leu Pro Pro 385 390 395 400 Thr Lys Val Glu Lys Cys Lys Ile Val Asp Ala Ile Ile Ser His Gly 405 410 415 Cys Phe Leu Arg Glu Cys Ser Val Gln His Ser Ile Val Gly Val Arg 420 425 430 Ser Arg Leu Glu Ser Gly Val Glu Leu Gln Asp Thr Met Met Met Gly 435 440 445 Ala Asp Tyr Tyr Gln Thr Glu Tyr Glu Ile Ala Ser Leu Val Ala Glu 450 455 460 Gly Lys Val Pro Ile Gly Val Gly Ala Asn Thr Lys Ile Arg Asn Cys 465 470 475 480 Ile Ile Asp Lys Asn Ala Lys Ile Gly Arg Asn Val Ile Ile Ala Asn 485 490 495 Thr Asp Gly Val Gln Glu Ala Asp Arg Ala Lys Glu Gly Phe Tyr Ile 500 505 510 Arg Ser Gly Ile Thr Val Thr Leu Lys Asn Ala Thr Ile Lys Asp Gly 515 520 525 Thr Val Ile 530 25521PRTCicer arietinum 25Met Asp Leu Ala Ile Gly Ser Asn Tyr Ala Ser Leu Arg Ser Ser Val 1 5 10 15 Phe Leu Gly Glu Thr Leu Lys Gly Asn Leu Ser Thr Lys Phe Leu Thr 20 25 30 Ser Pro Lys Phe Ser Gln Ile His Ile Asn Asn Leu Arg Ser Phe Asn 35 40 45 Pro Arg Asn Gly Ala Ser Tyr Ser Val Leu Thr Ser Gly Ile Asn Asp 50 55 60 Phe Glu Glu Ser Met Thr Phe His Glu Gly Pro Tyr Phe Asp Thr Pro 65 70 75 80 Lys Ala Asp Pro Lys Ser Val Ala Ser Ile Ile Leu Gly Gly Gly Ala 85 90 95 Gly Thr Arg Leu Phe Pro Leu Thr Ser Lys Arg Ala Lys Pro Ala Val 100 105 110 Pro Ile Gly Gly Cys Tyr Arg Leu Ile Asp Ile Pro Met Ser Asn Cys 115 120 125 Ile Asn Ser Gly Ile Arg Lys Ile Phe Ile Leu Thr Gln Phe Asn Ser 130 135 140 Phe Ser Leu Asn Arg His Leu Ser Arg Ser Tyr Asn Phe Gly Asn Val 145 150 155 160 Ser Thr Phe Gly Glu Gly Phe Val Glu Val Leu Ala Ala Thr Gln Thr 165 170 175 Ser Gly Glu Ala Gly Lys Lys Trp Phe Gln Gly Thr Ala Asp Ala Val 180 185 190 Arg Gln Phe Ile Trp Val Phe Glu Asp Ala Lys Thr Lys Asn Val Glu 195 200 205 His Ile Leu Ile Leu Ser Gly Asp His Leu Tyr Arg Met Asn Tyr Met 210 215 220 Asp Phe Val Gln Lys His Ile Asp Thr Asn Ala Asp Ile Thr Val Ser 225 230 235 240 Cys Ile Pro Met Asp Asp Ser Arg Ala Ser Asp Tyr Gly Leu Leu Lys 245 250 255 Ile Asp Gly Lys Gly Arg Ile Ile Gln Phe Ala Glu Lys Pro Lys Gly 260 265 270 Ser Glu Leu Lys Ala Met Arg Val Asp Thr Thr Leu Leu Gly Leu Ser 275 280 285 Pro Glu Glu Ala Lys Lys Gln Pro Tyr Ile Ala Ser Met Gly Val Tyr 290 295 300 Val Phe Arg Thr Glu Thr Leu Leu Lys Leu Leu Arg Ser Asn Cys Ser 305 310 315 320 Thr Cys Asn Asp Phe Gly Ser Glu Ile Ile Pro Ser Ala Val Asn Asp 325 330 335 Asp His Asn Val Gln Ala Tyr Leu Phe Asn Asp Tyr Trp Glu Asp Ile 340 345 350 Gly Thr Ile Lys Ser Phe Phe Asp Ala Asn Leu Ala Leu Thr Asp Gln 355 360 365 Pro Pro Lys Phe Gln Phe Tyr Asp Pro Asn Thr Pro Phe Tyr Thr Phe 370 375 380 Pro Arg Phe Leu Pro Pro Thr Lys Val Glu Lys Cys Lys Ile Val Asp 385 390 395 400 Ala Ile Ile Ser His Gly Cys Phe Leu Arg Glu Cys Ser Val Gln His 405 410 415 Ser Ile Val Gly Ile Arg Ser Arg Leu Glu Ser Gly Val Glu Leu Gln 420 425 430 Asp Thr Met Met Met Gly Ala Asp Tyr Tyr Gln Thr Glu Ser Glu Ile 435 440 445 Ala Ser Leu Leu Ala Glu Gly Lys Val Pro Val Gly Val Gly Glu Asn 450 455 460 Thr Lys Ile Arg Asn Cys Ile Ile Asp Lys Asn Ala Arg Ile Gly Arg 465 470 475 480 Asn Val Ile Ile Thr Asn Ala Asp Gly Val Glu Glu Ala Asp Arg Thr 485 490 495 Lys Glu Gly Phe Tyr Ile Arg Ser Gly Ile Thr Ala Ile Leu Lys Asn 500 505 510 Ala Thr Ile Lys Asp Gly Thr Val Ile 515 520 26531PRTGlycine maxmisc_feature(404)..(404)Xaa can be any naturally occurring amino acid 26Met Asp Ser Ala Cys Ala Thr Leu Asn Gly Arg His Leu Ala Lys Val 1 5 10 15 Ser Glu Gly Ile Gly Arg Asn Arg Thr Ser Gly Phe Trp Gly Glu Ser 20 25 30 Thr Arg Gly Ser Val Asn Thr Lys Arg Phe Leu Ser Val Gln Ser Cys 35 40 45 Lys Thr Ser Arg Thr Asn Arg Asn

Leu Arg Asn Ser Lys Pro Gly Ser 50 55 60 Gly Ile Ala Arg Ala Val Leu Thr Ser Asp Ile Asp Glu Asp Ser Met 65 70 75 80 Ala Phe Gln Gly Val Pro Thr Phe Glu Lys Pro Glu Val Asp Pro Lys 85 90 95 Ser Val Ala Ser Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Phe 100 105 110 Pro Leu Thr Gly Arg Arg Ala Lys Pro Ala Val Pro Ile Gly Gly Cys 115 120 125 Tyr Arg Leu Ile Asp Ile Pro Met Ser Asn Cys Ile Asn Ser Gly Ile 130 135 140 Arg Lys Ile Phe Ile Leu Thr Gln Phe Asn Ser Phe Ser Leu Asn Arg 145 150 155 160 His Leu Ser Arg Ala Tyr Ser Phe Gly Asn Gly Met Thr Phe Gly Asp 165 170 175 Gly Phe Val Glu Val Leu Ala Ala Thr Gln Thr Pro Gly Glu Ala Gly 180 185 190 Lys Lys Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Phe Ile Trp 195 200 205 Val Phe Glu Asp Ala Lys Asn Lys Asn Val Glu His Ile Leu Ile Leu 210 215 220 Ser Gly Asp His Leu Tyr Arg Met Asp Tyr Met Asp Phe Val Gln Arg 225 230 235 240 His Val Asp Thr Asn Ala Asp Ile Thr Val Ser Cys Val Pro Met Asp 245 250 255 Asp Ser Arg Ala Ser Asp Tyr Gly Leu Met Lys Ile Asp Lys Thr Gly 260 265 270 Arg Ile Ile Gln Phe Ala Glu Lys Pro Lys Gly Ser Asp Leu Lys Ala 275 280 285 Met Arg Val Asp Thr Thr Leu Leu Gly Leu Leu Pro Gln Glu Ala Glu 290 295 300 Lys His Pro Tyr Ile Ala Ser Met Gly Val Tyr Val Phe Arg Thr Glu 305 310 315 320 Thr Leu Leu Gln Leu Leu Arg Trp Lys Gly Ser Ser Cys Asn Asp Phe 325 330 335 Gly Ser Glu Ile Ile Pro Ser Ala Val Asn Glu His Asn Val Gln Ala 340 345 350 Tyr Leu Phe Asn Asp Tyr Trp Glu Asp Ile Gly Thr Ile Lys Ser Phe 355 360 365 Phe Asp Ala Asn Leu Ala Leu Thr Glu Gln Pro Pro Lys Phe Glu Phe 370 375 380 Tyr Asp Pro Lys Thr Pro Phe Phe Thr Ser Pro Arg Phe Leu Pro Pro 385 390 395 400 Thr Lys Val Xaa Lys Cys Lys Ile Val Asp Ala Ile Ile Ser His Gly 405 410 415 Cys Phe Leu Arg Glu Cys Ser Val Gln His Ser Ile Val Gly Val Arg 420 425 430 Ser Arg Leu Glu Ser Gly Val Glu Leu Gln Asp Thr Met Met Met Gly 435 440 445 Ala Asp Tyr Tyr Gln Thr Glu Tyr Glu Ile Ala Ser Leu Val Ala Glu 450 455 460 Gly Lys Val Pro Ile Gly Val Gly Ala Asn Thr Lys Ile Arg Asn Cys 465 470 475 480 Ile Ile Asp Lys Asn Ala Lys Ile Gly Arg Asn Val Ile Ile Ala Asn 485 490 495 Thr Asp Gly Val Gln Glu Ala Asp Arg Ala Lys Glu Gly Phe Tyr Ile 500 505 510 Arg Ser Gly Ile Thr Val Thr Leu Lys Asn Ala Thr Ile Lys Asp Gly 515 520 525 Thr Val Ile 530 271886DNAGlycine maxCDS(80)..(1627) 27gcacgagtta aagagagtca caagccaatt ctgaaccaca cactcactta tttgtttctt 60tcaactcact cacagagta atg gca tcc atg gcg gct ata ggt tct ctg aat 112 Met Ala Ser Met Ala Ala Ile Gly Ser Leu Asn 1 5 10 gtt cct tgt tct gct tct tcg cgt tca tcg aat gtg gga aga aaa agc 160Val Pro Cys Ser Ala Ser Ser Arg Ser Ser Asn Val Gly Arg Lys Ser 15 20 25 ttt cca cgc agc ctt tca ttc tct gca tca caa ctt tgt gga gac aag 208Phe Pro Arg Ser Leu Ser Phe Ser Ala Ser Gln Leu Cys Gly Asp Lys 30 35 40 att cac aca gat tca gtt tca ttc gca cca aaa atc ggt cgc aat cct 256Ile His Thr Asp Ser Val Ser Phe Ala Pro Lys Ile Gly Arg Asn Pro 45 50 55 gta att gtt acc cct aaa gca gtt tct gat tcc caa aac tcc caa acc 304Val Ile Val Thr Pro Lys Ala Val Ser Asp Ser Gln Asn Ser Gln Thr 60 65 70 75 tgt ctt gat ccc gat gct agc aga agt gtg ctt ggc att ata ctt gga 352Cys Leu Asp Pro Asp Ala Ser Arg Ser Val Leu Gly Ile Ile Leu Gly 80 85 90 ggt ggt gct ggg act cgt ctt tat cca ctg acc aag aag agg gca aag 400Gly Gly Ala Gly Thr Arg Leu Tyr Pro Leu Thr Lys Lys Arg Ala Lys 95 100 105 cca gct gtt cct ctt gga gct aac tat agg cta att gac att cct gtt 448Pro Ala Val Pro Leu Gly Ala Asn Tyr Arg Leu Ile Asp Ile Pro Val 110 115 120 agc aac tgc ttg aat agc aac gtc tcc aag atc tat gtt ctc act caa 496Ser Asn Cys Leu Asn Ser Asn Val Ser Lys Ile Tyr Val Leu Thr Gln 125 130 135 ttc aat tcc gcc tcg tta aac cga cac ctt tct cgt gct tat gca agc 544Phe Asn Ser Ala Ser Leu Asn Arg His Leu Ser Arg Ala Tyr Ala Ser 140 145 150 155 aac atg ggt ggc tac aaa aat gag gga ttt gtt gag gtt ctt gct gct 592Asn Met Gly Gly Tyr Lys Asn Glu Gly Phe Val Glu Val Leu Ala Ala 160 165 170 cag cag agt cct gag aat cct aat tgg ttc cag ggt act gca gat gct 640Gln Gln Ser Pro Glu Asn Pro Asn Trp Phe Gln Gly Thr Ala Asp Ala 175 180 185 gtc agg cag tat ttg tgg ctt ttt gaa gag cac aat gtt ttg gaa ttc 688Val Arg Gln Tyr Leu Trp Leu Phe Glu Glu His Asn Val Leu Glu Phe 190 195 200 ttg gtt ctg gct ggt gac cat ttg tat cga atg gat tac gag aaa ttt 736Leu Val Leu Ala Gly Asp His Leu Tyr Arg Met Asp Tyr Glu Lys Phe 205 210 215 atc caa gcg cat agg gaa act gat gct gat atc act gtg gct gca ttg 784Ile Gln Ala His Arg Glu Thr Asp Ala Asp Ile Thr Val Ala Ala Leu 220 225 230 235 cca atg gat gaa aag cgt gcc act gca ttt ggc ctg atg aag att gat 832Pro Met Asp Glu Lys Arg Ala Thr Ala Phe Gly Leu Met Lys Ile Asp 240 245 250 gaa gag ggg cgt ata att gaa ttc gcc gaa aag cca aaa gga gaa cag 880Glu Glu Gly Arg Ile Ile Glu Phe Ala Glu Lys Pro Lys Gly Glu Gln 255 260 265 ttg aaa gct atg aag gtt gat act aca att ttg ggt ctt gat gac gag 928Leu Lys Ala Met Lys Val Asp Thr Thr Ile Leu Gly Leu Asp Asp Glu 270 275 280 aga gca aag gaa ttg cct tat att gct agc atg ggt ata tat gtt gtt 976Arg Ala Lys Glu Leu Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val Val 285 290 295 agc aaa aac gtg atg tta gac ctg ctc cgt gag aag ttt cct ggt gca 1024Ser Lys Asn Val Met Leu Asp Leu Leu Arg Glu Lys Phe Pro Gly Ala 300 305 310 315 aat gac ttt ggg agt gaa gtg att cct ggt gct act tct att gga atg 1072Asn Asp Phe Gly Ser Glu Val Ile Pro Gly Ala Thr Ser Ile Gly Met 320 325 330 agg gtg caa gct tac ttg tat gat ggc tac tgg gaa gac att ggt aca 1120Arg Val Gln Ala Tyr Leu Tyr Asp Gly Tyr Trp Glu Asp Ile Gly Thr 335 340 345 att gag gct ttc tat aat gca aat ctg gga atc acc aaa aag cct gtg 1168Ile Glu Ala Phe Tyr Asn Ala Asn Leu Gly Ile Thr Lys Lys Pro Val 350 355 360 cct gac ttc agt ttc tat gat cgt tca tct cca atc tac acc caa cca 1216Pro Asp Phe Ser Phe Tyr Asp Arg Ser Ser Pro Ile Tyr Thr Gln Pro 365 370 375 cga tat ttg cct ccc tct aag atg ctt gat gct gat gtc act gat agt 1264Arg Tyr Leu Pro Pro Ser Lys Met Leu Asp Ala Asp Val Thr Asp Ser 380 385 390 395 gtt att ggt gaa gga tgt gtg att aag aac tgc aaa att cac cat tct 1312Val Ile Gly Glu Gly Cys Val Ile Lys Asn Cys Lys Ile His His Ser 400 405 410 gtg gtt ggg ctg cga tct tgc ata tca gaa ggt gca att att gaa gac 1360Val Val Gly Leu Arg Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp 415 420 425 acg tta tta atg ggg gca gat tat tac gag acg gag gct gat aag agg 1408Thr Leu Leu Met Gly Ala Asp Tyr Tyr Glu Thr Glu Ala Asp Lys Arg 430 435 440 ttt ctg gct gct aaa ggc agt gtt cca att ggt ata ggc agg aac tct 1456Phe Leu Ala Ala Lys Gly Ser Val Pro Ile Gly Ile Gly Arg Asn Ser 445 450 455 cat atc aaa agg gca att atc gac aag aat gct cga att ggg gaa aat 1504His Ile Lys Arg Ala Ile Ile Asp Lys Asn Ala Arg Ile Gly Glu Asn 460 465 470 475 gtc aag att att aac agt gac aat gtc caa gaa gct gca agg gaa aca 1552Val Lys Ile Ile Asn Ser Asp Asn Val Gln Glu Ala Ala Arg Glu Thr 480 485 490 gat ggg tat ttc ata aaa agt ggg att gtc aca gta atc aag gat gct 1600Asp Gly Tyr Phe Ile Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala 495 500 505 tta att cct agt gga aca gtc atc taa acaccaccac caccccaaaa 1647Leu Ile Pro Ser Gly Thr Val Ile 510 515 aatttcttgt accccaaatc ctaatggtga ctgcaaagct cattaccacc gctggagagt 1707ttatcaagct atgcttcctc gtctaagata ggcttttgtg tttcatgata tttatttttg 1767ggcagtggct tgtaaattat agcgggagag aaggcccgct atgagcaatc acgctgtaaa 1827gttcattatt caattgaata aaagtttctt cgtttcgtac taaaaaaaaa aaaaaaaaa 188628515PRTGlycine max 28Met Ala Ser Met Ala Ala Ile Gly Ser Leu Asn Val Pro Cys Ser Ala 1 5 10 15 Ser Ser Arg Ser Ser Asn Val Gly Arg Lys Ser Phe Pro Arg Ser Leu 20 25 30 Ser Phe Ser Ala Ser Gln Leu Cys Gly Asp Lys Ile His Thr Asp Ser 35 40 45 Val Ser Phe Ala Pro Lys Ile Gly Arg Asn Pro Val Ile Val Thr Pro 50 55 60 Lys Ala Val Ser Asp Ser Gln Asn Ser Gln Thr Cys Leu Asp Pro Asp 65 70 75 80 Ala Ser Arg Ser Val Leu Gly Ile Ile Leu Gly Gly Gly Ala Gly Thr 85 90 95 Arg Leu Tyr Pro Leu Thr Lys Lys Arg Ala Lys Pro Ala Val Pro Leu 100 105 110 Gly Ala Asn Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Leu Asn 115 120 125 Ser Asn Val Ser Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser 130 135 140 Leu Asn Arg His Leu Ser Arg Ala Tyr Ala Ser Asn Met Gly Gly Tyr 145 150 155 160 Lys Asn Glu Gly Phe Val Glu Val Leu Ala Ala Gln Gln Ser Pro Glu 165 170 175 Asn Pro Asn Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu 180 185 190 Trp Leu Phe Glu Glu His Asn Val Leu Glu Phe Leu Val Leu Ala Gly 195 200 205 Asp His Leu Tyr Arg Met Asp Tyr Glu Lys Phe Ile Gln Ala His Arg 210 215 220 Glu Thr Asp Ala Asp Ile Thr Val Ala Ala Leu Pro Met Asp Glu Lys 225 230 235 240 Arg Ala Thr Ala Phe Gly Leu Met Lys Ile Asp Glu Glu Gly Arg Ile 245 250 255 Ile Glu Phe Ala Glu Lys Pro Lys Gly Glu Gln Leu Lys Ala Met Lys 260 265 270 Val Asp Thr Thr Ile Leu Gly Leu Asp Asp Glu Arg Ala Lys Glu Leu 275 280 285 Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val Val Ser Lys Asn Val Met 290 295 300 Leu Asp Leu Leu Arg Glu Lys Phe Pro Gly Ala Asn Asp Phe Gly Ser 305 310 315 320 Glu Val Ile Pro Gly Ala Thr Ser Ile Gly Met Arg Val Gln Ala Tyr 325 330 335 Leu Tyr Asp Gly Tyr Trp Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr 340 345 350 Asn Ala Asn Leu Gly Ile Thr Lys Lys Pro Val Pro Asp Phe Ser Phe 355 360 365 Tyr Asp Arg Ser Ser Pro Ile Tyr Thr Gln Pro Arg Tyr Leu Pro Pro 370 375 380 Ser Lys Met Leu Asp Ala Asp Val Thr Asp Ser Val Ile Gly Glu Gly 385 390 395 400 Cys Val Ile Lys Asn Cys Lys Ile His His Ser Val Val Gly Leu Arg 405 410 415 Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp Thr Leu Leu Met Gly 420 425 430 Ala Asp Tyr Tyr Glu Thr Glu Ala Asp Lys Arg Phe Leu Ala Ala Lys 435 440 445 Gly Ser Val Pro Ile Gly Ile Gly Arg Asn Ser His Ile Lys Arg Ala 450 455 460 Ile Ile Asp Lys Asn Ala Arg Ile Gly Glu Asn Val Lys Ile Ile Asn 465 470 475 480 Ser Asp Asn Val Gln Glu Ala Ala Arg Glu Thr Asp Gly Tyr Phe Ile 485 490 495 Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala Leu Ile Pro Ser Gly 500 505 510 Thr Val Ile 515 291856DNAGlycine maxCDS(47)..(1594) 29ctttcaaccg agacactcac aagtcacaac acacacacac atagca atg gca tct 55 Met Ala Ser 1 atg gcg gct ata ggt tct ctc aat gtt cca cgt tct gct tct tcc cgt 103Met Ala Ala Ile Gly Ser Leu Asn Val Pro Arg Ser Ala Ser Ser Arg 5 10 15 tca tcc ttt gtg gga aga aaa agc gtt cca cgc agc ctt tcc ttc tct 151Ser Ser Phe Val Gly Arg Lys Ser Val Pro Arg Ser Leu Ser Phe Ser 20 25 30 35 gca tca caa ctt tgt gga gac aag att ccc aca gat tca gtt tta ttg 199Ala Ser Gln Leu Cys Gly Asp Lys Ile Pro Thr Asp Ser Val Leu Leu 40 45 50 gca cca aaa ata ggt cgc agt cca gtt atc gtt act cct aaa gca gtt 247Ala Pro Lys Ile Gly Arg Ser Pro Val Ile Val Thr Pro Lys Ala Val 55 60 65 tct gat tcc caa aac tca caa acg tgc ctt gat ccc gat gct agc aga 295Ser Asp Ser Gln Asn Ser Gln Thr Cys Leu Asp Pro Asp Ala Ser Arg 70 75 80 agt gtg ctt ggc att ata ctt gga ggt ggt gct ggg act cgg ctt tat 343Ser Val Leu Gly Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Tyr 85 90 95 cca ctc acc aag aag agg gca aag cca gct gtt cct ctt gga gct aac 391Pro Leu Thr Lys Lys Arg Ala Lys Pro Ala Val Pro Leu Gly Ala Asn 100 105 110 115 tat agg ctt att gat att cct gtt agc aac tgc ttg aat agc aat gtc 439Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Leu Asn Ser Asn Val 120 125 130 tcc aag atc tat gtt ctc act caa ttc aat tct gcg tcg tta aac cga 487Ser Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser Leu Asn Arg 135 140 145 cac ctt tct cgt gct tat gca agc aac atg ggt ggc tac aaa aat gag 535His Leu Ser Arg Ala Tyr Ala Ser Asn Met Gly Gly Tyr Lys Asn Glu 150 155 160 gga ttt gtt gag gtt ctt gct gct cag cag agt cct gag aat cct aat 583Gly Phe Val Glu Val Leu Ala Ala Gln Gln Ser Pro Glu Asn Pro Asn 165 170 175 tgg ttc cag ggt act gca gat gct gtg agg cag tat ttg tgg ctt ttt 631Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu Trp Leu Phe 180 185 190

195 gaa gag cac aat gtt ttg gaa ttc ttg gtt ctg gct ggt gac cat ttg 679Glu Glu His Asn Val Leu Glu Phe Leu Val Leu Ala Gly Asp His Leu 200 205 210 tat cga atg gat tac gag aaa ttt atc caa gcg cat agg gaa act gat 727Tyr Arg Met Asp Tyr Glu Lys Phe Ile Gln Ala His Arg Glu Thr Asp 215 220 225 gct gat atc act gtg gct gca ttg cca atg gat gaa gcg cgt gcc act 775Ala Asp Ile Thr Val Ala Ala Leu Pro Met Asp Glu Ala Arg Ala Thr 230 235 240 gca ttt ggt ttg atg aag att gat gaa gag ggg cgt ata att gaa ttt 823Ala Phe Gly Leu Met Lys Ile Asp Glu Glu Gly Arg Ile Ile Glu Phe 245 250 255 gct gaa aag cca aaa gga gaa cag ttg aaa gct atg aag gtt gat act 871Ala Glu Lys Pro Lys Gly Glu Gln Leu Lys Ala Met Lys Val Asp Thr 260 265 270 275 aca att ttg ggt ctt gat gac gag aga gca aaa gaa atg cct tat att 919Thr Ile Leu Gly Leu Asp Asp Glu Arg Ala Lys Glu Met Pro Tyr Ile 280 285 290 gct agc atg ggt ata tat gtt gtt agc aaa aat gtg atg tta gac ctg 967Ala Ser Met Gly Ile Tyr Val Val Ser Lys Asn Val Met Leu Asp Leu 295 300 305 ctc cgt gag aag ttt cct ggt gca aat gac ttt ggg agt gaa gtg att 1015Leu Arg Glu Lys Phe Pro Gly Ala Asn Asp Phe Gly Ser Glu Val Ile 310 315 320 cct ggt gct act tct att gga atg aga gtg caa gct tac ttg tat gat 1063Pro Gly Ala Thr Ser Ile Gly Met Arg Val Gln Ala Tyr Leu Tyr Asp 325 330 335 ggc tac tgg gaa gac att ggt aca atc gag gct ttc tat aat gca aat 1111Gly Tyr Trp Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn 340 345 350 355 ctg gga atc acc aaa aag cct gtg cct gac ttc agt ttc tat gat cgt 1159Leu Gly Ile Thr Lys Lys Pro Val Pro Asp Phe Ser Phe Tyr Asp Arg 360 365 370 tca tct cca atc tac acc caa cca cga tat ttg cct ccc tct aag atg 1207Ser Ser Pro Ile Tyr Thr Gln Pro Arg Tyr Leu Pro Pro Ser Lys Met 375 380 385 ctt gat gct gat gtc act gat agt gtt att ggt gaa gga tgt gtg att 1255Leu Asp Ala Asp Val Thr Asp Ser Val Ile Gly Glu Gly Cys Val Ile 390 395 400 aag aac tgc aaa att cac cat tct gtg gtt ggg ctg cga tct tgc ata 1303Lys Asn Cys Lys Ile His His Ser Val Val Gly Leu Arg Ser Cys Ile 405 410 415 tca gaa ggt gca att att gaa gac acg tta tta atg ggg gca gat tat 1351Ser Glu Gly Ala Ile Ile Glu Asp Thr Leu Leu Met Gly Ala Asp Tyr 420 425 430 435 tac gag acg gag gct gat aag agg ttt ctg gct gcc aaa ggc agt gtt 1399Tyr Glu Thr Glu Ala Asp Lys Arg Phe Leu Ala Ala Lys Gly Ser Val 440 445 450 cca att ggt ata ggc agg aac tct cat atc aaa agg gca att att gac 1447Pro Ile Gly Ile Gly Arg Asn Ser His Ile Lys Arg Ala Ile Ile Asp 455 460 465 aag aat gct cga att ggg gaa aat gtc aag att att aac agt gac aat 1495Lys Asn Ala Arg Ile Gly Glu Asn Val Lys Ile Ile Asn Ser Asp Asn 470 475 480 gtc caa gaa gct gca agg gaa aca gat ggg tat ttc ata aaa agt ggg 1543Val Gln Glu Ala Ala Arg Glu Thr Asp Gly Tyr Phe Ile Lys Ser Gly 485 490 495 att gtc aca gta atc aag gat gct tta att cct agt gga aca gtc atc 1591Ile Val Thr Val Ile Lys Asp Ala Leu Ile Pro Ser Gly Thr Val Ile 500 505 510 515 taa acacaaccac ctccccaaaa aatttcttgt accccaaatc ctaatggtga 1644ctgcgaagct cattaccacc gcaggagagt ttatcaagct ctgcttccac gtctaagata 1704ggcttttgtg tttcatgata tatatttttg ggcagtggct tgtaaataat agcggaagag 1764aaggcccgct atgagcaatc acgctgtaaa gttcgttaat caattcaata aaacaagttt 1824ctttatttcg tactaaaaaa aaaaaaaaaa aa 185630515PRTGlycine max 30Met Ala Ser Met Ala Ala Ile Gly Ser Leu Asn Val Pro Arg Ser Ala 1 5 10 15 Ser Ser Arg Ser Ser Phe Val Gly Arg Lys Ser Val Pro Arg Ser Leu 20 25 30 Ser Phe Ser Ala Ser Gln Leu Cys Gly Asp Lys Ile Pro Thr Asp Ser 35 40 45 Val Leu Leu Ala Pro Lys Ile Gly Arg Ser Pro Val Ile Val Thr Pro 50 55 60 Lys Ala Val Ser Asp Ser Gln Asn Ser Gln Thr Cys Leu Asp Pro Asp 65 70 75 80 Ala Ser Arg Ser Val Leu Gly Ile Ile Leu Gly Gly Gly Ala Gly Thr 85 90 95 Arg Leu Tyr Pro Leu Thr Lys Lys Arg Ala Lys Pro Ala Val Pro Leu 100 105 110 Gly Ala Asn Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Leu Asn 115 120 125 Ser Asn Val Ser Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser 130 135 140 Leu Asn Arg His Leu Ser Arg Ala Tyr Ala Ser Asn Met Gly Gly Tyr 145 150 155 160 Lys Asn Glu Gly Phe Val Glu Val Leu Ala Ala Gln Gln Ser Pro Glu 165 170 175 Asn Pro Asn Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu 180 185 190 Trp Leu Phe Glu Glu His Asn Val Leu Glu Phe Leu Val Leu Ala Gly 195 200 205 Asp His Leu Tyr Arg Met Asp Tyr Glu Lys Phe Ile Gln Ala His Arg 210 215 220 Glu Thr Asp Ala Asp Ile Thr Val Ala Ala Leu Pro Met Asp Glu Ala 225 230 235 240 Arg Ala Thr Ala Phe Gly Leu Met Lys Ile Asp Glu Glu Gly Arg Ile 245 250 255 Ile Glu Phe Ala Glu Lys Pro Lys Gly Glu Gln Leu Lys Ala Met Lys 260 265 270 Val Asp Thr Thr Ile Leu Gly Leu Asp Asp Glu Arg Ala Lys Glu Met 275 280 285 Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val Val Ser Lys Asn Val Met 290 295 300 Leu Asp Leu Leu Arg Glu Lys Phe Pro Gly Ala Asn Asp Phe Gly Ser 305 310 315 320 Glu Val Ile Pro Gly Ala Thr Ser Ile Gly Met Arg Val Gln Ala Tyr 325 330 335 Leu Tyr Asp Gly Tyr Trp Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr 340 345 350 Asn Ala Asn Leu Gly Ile Thr Lys Lys Pro Val Pro Asp Phe Ser Phe 355 360 365 Tyr Asp Arg Ser Ser Pro Ile Tyr Thr Gln Pro Arg Tyr Leu Pro Pro 370 375 380 Ser Lys Met Leu Asp Ala Asp Val Thr Asp Ser Val Ile Gly Glu Gly 385 390 395 400 Cys Val Ile Lys Asn Cys Lys Ile His His Ser Val Val Gly Leu Arg 405 410 415 Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp Thr Leu Leu Met Gly 420 425 430 Ala Asp Tyr Tyr Glu Thr Glu Ala Asp Lys Arg Phe Leu Ala Ala Lys 435 440 445 Gly Ser Val Pro Ile Gly Ile Gly Arg Asn Ser His Ile Lys Arg Ala 450 455 460 Ile Ile Asp Lys Asn Ala Arg Ile Gly Glu Asn Val Lys Ile Ile Asn 465 470 475 480 Ser Asp Asn Val Gln Glu Ala Ala Arg Glu Thr Asp Gly Tyr Phe Ile 485 490 495 Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala Leu Ile Pro Ser Gly 500 505 510 Thr Val Ile 515 31515PRTPhaseolus vulgaris 31Met Ala Ser Met Ala Ser Ile Gly Ser Leu Asn Val Pro Cys Ser Ser 1 5 10 15 Ser Ser Ser Ser Ser Asn Gly Gly Arg Lys Ile Leu Pro Arg Ala Leu 20 25 30 Ser Phe Ser Ala Ser Gln Leu Tyr Gly Asp Lys Ile Ser Thr Asp Ser 35 40 45 Val Ser Val Ala Pro Lys Arg Val Arg Asn Pro Val Val Val Ser Pro 50 55 60 Lys Ala Val Ser Asp Ser Gln Asn Ser Gln Thr Cys Leu Asp Pro Asp 65 70 75 80 Ala Ser Lys Ser Val Leu Gly Ile Ile Leu Gly Gly Gly Ala Gly Thr 85 90 95 Arg Leu Tyr Pro Leu Thr Lys Lys Arg Ala Lys Pro Ala Val Pro Leu 100 105 110 Gly Ala Asn Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Leu Asn 115 120 125 Ser Asn Val Ser Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser 130 135 140 Leu Asn Arg His Leu Ser Arg Ala Tyr Ala Ser Asn Met Gly Gly Tyr 145 150 155 160 Lys Asn Glu Gly Phe Val Glu Val Leu Ala Ala Gln Gln Ser Pro Glu 165 170 175 Asn Pro Asn Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu 180 185 190 Trp Leu Phe Glu Glu His Asn Val Leu Glu Tyr Leu Val Leu Ala Gly 195 200 205 Asp His Leu Tyr Arg Met Asp Tyr Glu Lys Phe Ile Gln Val His Arg 210 215 220 Glu Ser Asp Ala Asp Ile Thr Val Ala Ala Leu Pro Met Asp Glu Asn 225 230 235 240 Arg Ala Thr Ala Phe Gly Leu Met Lys Ile Asp Glu Glu Gly Arg Ile 245 250 255 Ile Glu Phe Ala Glu Lys Pro Lys Gly Glu Gln Leu Lys Ala Met Lys 260 265 270 Val Asp Thr Thr Ile Phe Gly Leu Asp Asp Glu Arg Ala Lys Glu Met 275 280 285 Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val Val Ser Lys Asn Val Met 290 295 300 Leu Asn Leu Leu Arg Glu Lys Phe Pro Ala Ala Asn Asp Phe Gly Ser 305 310 315 320 Glu Val Ile Pro Gly Ala Thr Ser Ile Gly Leu Arg Val Gln Ala Tyr 325 330 335 Leu Tyr Asp Gly Tyr Trp Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr 340 345 350 Asn Ala Asn Leu Gly Ile Thr Lys Lys Pro Val Pro Asp Phe Ser Phe 355 360 365 Tyr Gly Arg Ser Ser Pro Ile Tyr Thr Gln Pro Arg Tyr Leu Pro Pro 370 375 380 Ser Lys Met Leu Asp Ala Asp Val Thr Asp Ser Val Ile Gly Glu Gly 385 390 395 400 Cys Val Ile Lys Asn Cys Lys Ile His His Ser Val Val Gly Leu Arg 405 410 415 Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp Thr Leu Leu Met Gly 420 425 430 Ala Asp Tyr Tyr Glu Thr Asp Ala Asp Lys Arg Phe Leu Ala Ala Lys 435 440 445 Gly Ser Val Pro Ile Gly Ile Gly Arg Asn Ser His Val Lys Arg Ala 450 455 460 Ile Ile Asp Lys Asn Ala Arg Ile Gly Glu Asn Val Lys Ile Leu Asn 465 470 475 480 Ser Asp Asn Val Gln Glu Ala Ala Arg Glu Thr Asp Gly Tyr Phe Ile 485 490 495 Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala Leu Ile Pro Ser Gly 500 505 510 Thr Val Ile 515 32515PRTGlycine max 32Met Ala Ser Met Ala Ala Ile Gly Ser Leu Asn Val Pro Arg Ser Ala 1 5 10 15 Ser Ser Arg Ser Ser Phe Val Gly Arg Lys Ser Val Pro Arg Ser Leu 20 25 30 Ser Phe Ser Ala Ser Gln Leu Cys Gly Asp Lys Ile Pro Thr Asp Ser 35 40 45 Val Leu Leu Ala Pro Lys Ile Gly Arg Ser Pro Val Ile Val Thr Pro 50 55 60 Lys Ala Val Ser Asp Ser Gln Asn Ser Gln Thr Cys Leu Asp Pro Asp 65 70 75 80 Ala Ser Arg Ser Val Leu Gly Ile Ile Leu Gly Gly Gly Ala Gly Thr 85 90 95 Arg Leu Tyr Pro Leu Thr Lys Lys Arg Ala Lys Pro Ala Val Pro Leu 100 105 110 Gly Ala Asn Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Leu Asn 115 120 125 Ser Asn Val Ser Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser 130 135 140 Leu Asn Arg His Leu Ser Arg Ala Tyr Ala Ser Asn Met Gly Gly Tyr 145 150 155 160 Lys Asn Glu Gly Phe Val Glu Val Leu Ala Ala Gln Gln Ser Pro Glu 165 170 175 Asn Pro Asn Trp Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu 180 185 190 Trp Leu Phe Glu Glu His Asn Val Leu Glu Phe Leu Val Leu Ala Gly 195 200 205 Asp His Leu Tyr Arg Met Asp Tyr Glu Lys Phe Ile Gln Ala His Arg 210 215 220 Glu Thr Asp Ala Asp Ile Thr Val Ala Ala Leu Pro Met Asp Glu Ala 225 230 235 240 Arg Ala Thr Ala Phe Gly Leu Met Lys Ile Asp Glu Glu Gly Arg Ile 245 250 255 Ile Glu Phe Ala Glu Lys Pro Lys Gly Glu Gln Leu Lys Ala Met Lys 260 265 270 Val Asp Thr Thr Ile Leu Gly Leu Asp Asp Glu Arg Ala Lys Glu Met 275 280 285 Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val Val Ser Lys Asn Val Met 290 295 300 Leu Asp Leu Leu Arg Glu Lys Phe Pro Gly Ala Asn Asp Phe Gly Ser 305 310 315 320 Glu Val Ile Pro Gly Ala Thr Ser Ile Gly Met Arg Val Gln Ala Tyr 325 330 335 Leu Tyr Asp Gly Tyr Trp Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr 340 345 350 Asn Ala Asn Leu Gly Ile Thr Lys Lys Pro Val Pro Asp Phe Ser Phe 355 360 365 Tyr Asp Arg Ser Ser Pro Ile Tyr Thr Gln Pro Arg Tyr Leu Pro Pro 370 375 380 Ser Lys Met Leu Asp Ala Asp Val Thr Asp Ser Val Ile Gly Glu Gly 385 390 395 400 Cys Val Ile Lys Asn Cys Lys Ile His His Ser Val Val Gly Leu Arg 405 410 415 Ser Cys Ile Ser Glu Gly Ala Ile Ile Glu Asp Thr Leu Leu Met Gly 420 425 430 Ala Asp Tyr Tyr Glu Thr Glu Ala Asp Lys Arg Phe Leu Ala Ala Lys 435 440 445 Gly Ser Val Pro Ile Gly Ile Gly Arg Asn Ser His Ile Lys Arg Ala 450 455 460 Ile Ile Asp Lys Asn Ala Arg Ile Gly Glu Asn Val Lys Ile Ile Asn 465 470 475 480 Ser Asp Asn Val Gln Glu Ala Ala Arg Glu Thr Asp Gly Tyr Phe Ile 485 490 495 Lys Ser Gly Ile Val Thr Val Ile Lys Asp Ala Leu Ile Pro Ser Gly 500 505 510 Thr Val Ile 515

Claims

1. An isolated polynucleotide comprising: (a) a first nucleotide sequence encoding a first polypeptide having phosphoglucomutase activity, wherein the amino acid sequence of the first polypeptide and the amino acid sequence of SEQ ID NO:8 have at least 95% identity based on the Clustal alignment method, (b) a second nucleotide sequence encoding a second polypeptide having phosphoglucomutase activity, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 85% identity based on the Clustal alignment method, or (c) the complement of the first or second nucleotide sequence.

2. The isolated polynucleotide of claim 1 wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 90% identity based on the Clustal alignment method.

3. The isolated polynucleotide of claim 1, wherein the amino acid sequence of the second polypeptide and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 95% identity based on the Clustal alignment method.

4. The isolated polynucleotide of claim 1, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:8, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

5. The isolated polynucleotide of claim 1, wherein the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:7, wherein the second nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

6. A recombinant DNA construct comprising the polynucleotide of claim 1 operably linked to a regulatory sequence.

7. A method for transforming a cell comprising transforming a cell with the polynucleotide of claim 1.

8. A cell comprising the recombinant DNA construct of claim 6.

9. A method for producing a transgenic plant comprising transforming a plant cell with the polynucleotide of claim 1 and regenerating a plant from the transformed plant cell.

10. A plant comprising the recombinant DNA construct of claim 6.

11. A seed comprising the recombinant DNA construct of claim 6.

12. An isolated polypeptide having phosphoglucomutase activity, wherein the polypeptide comprises: (a) a first amino acid sequence, wherein the first amino acid sequence and the amino acid sequence of SEQ ID NO:8 have at least 95% identity based on the Clustal alignment method, or (b) a second amino acid sequence, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 85% identity based on the Clustal alignment method.

13. The polypeptide of claim 12, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 90% identity based on the Clustal alignment method.

14. The polypeptide of claim 12, wherein the second amino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 95% identity based on the Clustal alignment method.

15. The polypeptide of claim 12, wherein the first amino acid sequence comprises the amino acid sequence of SEQ ID NO:8, and wherein the second amino acid sequence comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

16. A method of selecting an isolated polynucleotide that decreases the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a plant cell, the method comprising the steps of: (a) constructing the isolated polynucleotide comprising a nucleotide sequence of at least 30 contiguous nucleotides derived from the isolated polynucleotide of claim 1; (b) introducing the isolated polynucleotide into the plant cell; (c) measuring the level of the polypeptide in the plant cell containing the polynucleotide; and (d) comparing the level of the polypeptide in the plant cell containing the isolated polynucleotide with the level of the polypeptide in a plant cell that does not contain the isolated polynucleotide; and selecting the isolated polynucleotide that decreases the level of expression of the plastidic polypeptide having phosphoglucomutase activity in the plant cell.

17. The method of claim 16 wherein the isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3 and 7.

18. The method of claim 16 wherein the isolated polynucleotide comprising a nucleotide sequence of at least 30 contiguous nucleotides contains at least 541 nucleotides.

19. The method of claim 16 wherein the isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO:15.

20. A method of selecting an isolated polynucleotide that increases the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a plant cell, the method comprising the steps of: (a) constructing the isolated polynucleotide of claim 1; (b) introducing the isolated polynucleotide into the plant cell; (c) measuring the level of the polypeptide in the plant cell containing the polynucleotide; (d) comparing the level of the polypeptide in the plant cell containing the isolated polynucleotide with the level of the polypeptide in a plant cell that does not contain the polynucleotide; and selecting the isolated polynucleotide that increases the level of expression of the plastidic polypeptide having phosphoglucomutase activity in the plant cell.

21. A method for positive selection of a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct of claim 6; and (b) growing the transformed host cell under conditions which allow expression of a polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

22. The method of claim 21 wherein the host cell is a plant.

23. The method of claim 21 wherein the plant cell is a monocot.

24. The method of claim 21 wherein the plant cell is a dicot.

25. A method of increasing the level of expression of a plastidic polypeptide having phosphoglucomutase activity in a host cell comprising: (a) transforming a host cell with the recombinant DNA construct of claim 6; and (b) growing the transformed host cell from step (a) under conditions that are suitable for expression of the recombinant DNA construct; and (c) selecting a transformed cell wherein expression of the recombinant DNA construct results in production of higher levels of a plastidic polypeptide having phosphoglucomutase activity in the transformed host cell.

26. A method for suppressing the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity in a transgenic plant, wherein the method comprises: (a) transforming a plant cell with a fragment of the isolated polynucleotide of claim 1; (b) regenerating a transgenic plant from the transformed plant cell of (a); and (c) selecting a transgenic plant wherein the level of expression of a gene encoding a plastidic polypeptide having phosphoglucomutase activity has been suppressed.

27. A recombinant DNA construct comprising: (a) all or part of the nucleotide sequence set forth in SEQ ID NO:7 or SEQ ID NO:15; (b) the complement of (a); wherein (a) or (b) is useful in co-suppression or antisense suppression of endogenous phosphoglucomutase activity in a transgenic plant.

28. A method for producing transgenic seed, the method comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 27; (b) regenerating a transgenic plant from the transformed plant cell of (a); and (c) selecting a transgenic plant that produces a transgenic seed having an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% as compared to seed obtained from a non-transgenic plant.

29. The method of claim 28 wherein the increase in the combined oil and protein content is at least 1.8%.

30. The method of claim 28 wherein the increase in the combined oil and protein content is at least 2.0%.

31. A method for producing transgenic seed, the method comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 27; (b) regenerating a transgenic plant from the transformed plant cell of (a); and (c) selecting a transgenic plant that produces a transgenic seed having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to seed obtained from a non-transgenic plant.

32. The method of claim 31 wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 1.6%.

33. The method of claim 31 wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 1.8%.

34. The method of claim 31 wherein the transgenic seed differs from a non-transgenic seed by having an increase in the combined oil and protein content of at least 2.0%.

35. A method for producing defatted meal from transgenic seed, the method comprising: (a) transforming a plant cell with the recombinant DNA construct of claim 27; (b) regenerating a transgenic plant from the transformed plant cell of (a); and (c) selecting a transgenic plant that produces a transgenic seed wherein said seed is processed into defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% as compared to defatted meal obtained from seed of a non-transgenic plant.

36. The method of claim 35 wherein the defatted meal of the transgenic seed has a sucrose to raffinose family oligosaccharide ratio of 1.0 or less as compared to the sucrose to raffinose family oligosaccharide ratio of defatted meal obtained from a non-transgenic seed.

37. The method of any one of claims 28-36 wherein the transgenic seed is obtained from a transgenic dicot plant comprising in its genome the recombinant construct.

38. The method of claim 37 wherein the dicot plant is selected from the group consisting of Arabidopsis, soybean, oilseed Brassica, peanut, sunflower, safflower, cotton, tobacco, tomato, potato, and cocoa.

39. The method of claim 37 wherein the dicot plant is soybean.

40. A transgenic seed comprising the recombinant DNA construct of claim 27 in its genome wherein said transgenic seed has an increase in the combined oil and protein content of at least 1.6% and a decrease in the sucrose content of at least 25% when compared to a non-transgenic seed.

41. The transgenic seed of claim 40 wherein the increase in the combined oil and protein content is at least 1.8%.

42. The transgenic seed of claim 40 wherein the increase in the combined oil and protein content is at least 2.0%.

43. A transgenic seed comprising the recombinant DNA construct of claim 27 in its genome wherein said transgenic seed has a sucrose to raffinose family oligosaccharide ratio of 1.0 or less when compared to a non-transgenic seed.

44. The transgenic seed of claim 43 wherein the transgenic seed has an increase in the combined oil and protein content of at least 1.6% when compared to a non-transgenic seed.

45. The transgenic seed of claim 43 wherein the transgenic seed has an increase in the combined oil and protein content of at least 1.8% when compared to a non-transgenic seed.

46. The transgenic seed of claim 43 wherein the transgenic seed has an increase in the combined oil and protein content of at least 2.0% when compared to a non-transgenic seed.

47. Transgenic seed comprising the recombinant construct of claim 27 in its genome wherein said transgenic seed is processed to make defatted meal having an increase in the combined oil and protein content of at least 5% and a decrease in the sucrose content of at least 25% when compared to defatted meal obtained from a non-transgenic seed.

48. The transgenic seed of claim 47 wherein the defatted meal of the transgenic seed differs from the defatted meal of a non-transgenic seed by having a sucrose to raffinose family oligosaccharide ratio of 1.0 or less.

49. The transgenic seed of any one of claims 40-48 wherein the seed is a soybean seed.