Plant Galactinol Synthase Homologs

Abstract

Isolated nucleic acid fragments encoding galactinol synthase are disclosed. Recombinant DNA construct(s) for use in altering expression of endogenous genes encoding galactinol synthase are also disclosed.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60/581,851, filed Jun. 22, 2004, the entire content of which is 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 polynucleotides comprising nucleic acid fragments encoding galactinol synthase homologs in plants and seeds wherein all or part of such isolated polynucleotides can be used to down-regulate expression of endogenous genes encoding galactinol synthase.

BACKGROUND OF THE INVENTION

[0003] Raffinose saccharides are a group of D-galactose-containing oligosaccharide derivatives of sucrose that are widely distributed in plants. Raffinose saccharides are characterized by the general formula: [O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n-.alpha.-glucopyranosyl-(1- .fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4 are known respectively as sucrose, raffinose, stachyose, verbascose, and ajugose. A set of galactosyltransferases is involved in the biosynthesis of raffinose saccharides. Galactinol synthase (EC 2.4.1.123) catalyzes the synthesis of galactinol (O-.alpha.-D-gal-actopyranosyl-[1.fwdarw.1]-L-myo-inositol) from UDP-D-Gal and myo-inositol. Raffinose and stachyose are then synthesized by addition of Gal units from galactinol to sucrose and raffinose, respectively. These reversible reactions are mediated by raffinose synthase (EC 2.4.1.82) and stachyose synthase (EC 2.4.1.67). Transfer of a further Gal residue from galactinol to stachyose gives verbascose.

[0004] Extensive botanical surveys of the occurrence of raffinose saccharides have been reported in the scientific literature [see Dey (1985) in Biochemistry of Storage Carbohydrates in Green Plants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp. 53-129]. Raffinose saccharides are thought to be second only to sucrose with respect to abundance among the nonstructural carbohydrates in the plant kingdom. In fact, raffinose saccharides may be ubiquitous, at least among higher plants. Raffinose saccharides accumulate in significant quantities in the edible portion of many economically significant crop species. Examples include soybean (Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossypium hirsutum L.), canola (Brassica sp.) and all of the major edible leguminous crops including beans (Phaseolus sp.), chick pea (Cicer arietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas (Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).

[0005] Although abundant in many species, raffinose saccharides are an obstacle to the efficient utilization of some economically important crop species. Raffinose saccharides are not digested directly by animals, primarily because alpha-galactosidase is not present in the intestinal mucosa [Gitzelmann et al. (1965) Pediatrics 36:231-236; Rutloff et al. (1967) Nahrung 11:39-46]. However, microflora in the lower gut are readily able to ferment the raffinose saccharides resulting in an acidification of the gut and production of carbon dioxide, methane and hydrogen gases [Murphy et al. (1972) J. Agr. Food. Chem. 20:813-817; Cristofaro et al. (1974) in Sugars in Nutrition, H. L. Sipple and K. W. McNutt, Eds. Academic Press, New York, Chap. 20, 313-335; Reddy et al. (1980) J. Food Science 45:1161-1164]. The resulting flatulence can severely limit the use of leguminous plants in animal, particularly human, diets. It is unfortunate that the presence of raffinose saccharides restricts the use of legumes in human diets because many of these species are otherwise excellent sources of protein and soluble fiber. Varieties of edible beans free of raffinose saccharides would be more valuable for human diets and would more fully use the desirable nutritional qualities of edible leguminous plants.

[0006] The biosynthesis of raffinose saccharides has been well characterized [see Dey (1985) in Biochemistry of Storage Carbohydrates in Green Plants, P. M. Dey and R. A. Dixon, Eds. Academic Press, London, pp. 53-129]. The committed reaction of raffinose saccharide biosynthesis involves the synthesis of galactinol from UDP-galactose and myo-inositol. The enzyme that catalyzes this reaction is galactinol synthase (inositol 1-alpha-galactosyltransferase; EC 2.4.1.123). Synthesis of raffinose and higher homologues in the raffinose saccharide family from sucrose is thought to be catalyzed by distinct galactosyltransferases (for example, raffinose synthase and stachyose synthase). Studies in many species suggest that galactinol synthase is the key enzyme controlling the flux of reduced carbon into the biosynthesis of raffinose saccharides [Handley et al. (1983) J. Amer. Soc. Hort. Sci. 108:600-605; Saravitz, et al. (1987) Plant Physiol. 83:185-189].

[0007] Related galactinol synthase genes already known in the art include sequences disclosed in WO 01/77306 and in U.S. Pat. No. 5,648,210, Kerr et al. (the contents of which are hereby incorporated by reference), and Sprenger and Keller (2000) Plant J 21:249-258. Presumably related sequences are also disclosed in WO 98/50553.

[0008] There is a great deal of interest in identifying the genes that encode proteins involved in raffinose saccharides in plants. Specifically, the galactinol synthase gene may be used to alter galactinol synthesis and modulate the level of raffinose saccharides in plant cells. Accordingly, the availability of nucleic acid sequences encoding all or a portion of a galactinol synthase would facilitate studies to better understand raffinose synthesis in plants, and provide genetic tools to alter raffinose saccharide synthesis to enhance the nutritional qualities of many edible leguminous plants.

SUMMARY OF THE INVENTION

[0009] In a first embodiment, the invention concerns an isolated polynucleotide comprising:

[0010] (a) a nucleotide sequence encoding a polypeptide having galactinol synthase activity, wherein the polypeptide has an amino acid sequence of at least 85% identity, when compared to one of SEQ ID NO: 2 or 4 or 95% identity when compared to one of SEQ ID NO:6, based on the Clustal V method of alignment,

[0011] (b) all or part of the isolated polynucleotide comprising (a) for use in co-suppression or antisense suppression of endogenous nucleic acid sequences encoding polypeptides having galactinol synthase activity, or

[0012] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

[0013] In a second embodiment, the instant invention concerns a recombinant DNA construct comprising any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence, and a cell, a plant, and a seed comprising the recombinant DNA construct.

[0014] In a third embodiment, the present invention includes a vector comprising any of the isolated polynucleotides of the present invention.

[0015] In a fourth embodiment, the present invention concerns a method for transforming a cell comprising transforming a cell with any of the isolated polynucleotides of the present invention. The cell transformed by this method is also included. Advantageously, the cell is eukaryotic, e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

[0016] In a fifth embodiment, the present invention includes 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 plant from the transformed plant cell. The invention is also directed to the transgenic plant produced by this method, and seed obtained from this transgenic plant.

[0017] In a sixth embodiment, the present invention concerns an isolated polypeptide having galactinol synthase activity, wherein the polypeptide has an amino acid sequence of at least 85%, 90%, or 95% identity, based on the Clustal V method of alignment, when compared to one of SEQ ID NO: 2 or 4 and wherein the polypeptide has an amino acid sequence of at least 95% identity, based on the Clustal V method of alignment, when compared to SEQ ID NO:6.

[0018] In a seventh embodiment, the present invention concerns a method for isolating a polypeptide having galactinol synthase activity comprising isolating the polypeptide from a cell or culture medium of the cell, wherein the cell comprises a recombinant DNA construct comprising a polynucleotide of the invention operably linked to at least one regulatory sequence.

[0019] In an eighth embodiment, this invention concerns a method for selecting a transformed cell comprising: (a) transforming a host cell with the recombinant DNA construct or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably a plant cell, under conditions that allow expression of the galactinol synthase polynucleotide in an amount sufficient to complement a null mutant in order to provide a positive selection means.

[0020] In a ninth embodiment, this invention relates to a method of reducing the raffinose saccharide content of soybean seeds by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% or any integer percentage in between 30% to 100%.

[0021] In a tenth embodiment, this invention relates to a method of reducing the total stachyose content by at least 36%, 40%, 50%, 60%, 70%, 80%, 90%; 95% or 100% or any integer percentage between 36% to 100%.

[0022] In another embodiment, this invention relates to a method of reducing the level of at least one raffinose saccharide in soybean.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0023] The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.

[0024] FIG. 1 shows a comparison of the amino acid sequence alignment between the galactinol synthase encoded by the nucleotide sequences derived from soybean clones sdp3c.pk013.c9:fis, srr3c.pk003.h12:fis and srr3c.pk001.120:fis (SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, respectively) and the galactinol synthase from Pisum sativum (NCBI GenBank Identifier (GI) No. 5541885; SEQ ID NO:7), Arabidopsis thaliana (NCBI GenBank Identifier (GI) No. 15223567; SEQ ID NO:8) and Glycine max (NCBI GenBank Identifier (GI) No. 32345694; SEQ ID NO:9). Amino acids which are conserved among all sequences are indicated with an asterisk (*) below the conserved residue. The program to maximize alignment of the sequences uses dashes.

[0025] FIG. 2 shows vector pJMS08.

[0026] FIG. 3 shows vector pJMS10.

[0027] FIG. 4 shows a typical carbohydrate profile of transgenic somatic embryos co-suppressing galactinol synthase.

[0028] FIG. 5 shows a typical carbohydrate profile of a wild type soybean somatic embryo.

[0029] FIG. 6 shows vector pG4G.

[0030] FIG. 7 shows vector SH50.

[0031] FIG. 8 shows a TLC analysis of somatic embryos containing the PM29 driven recombinant expression construct described in Example 11.

[0032] FIG. 9 shows a TLC analysis of mature soybean seeds containing the PM29 driven recombinant expression construct described in Example 11.

[0033] FIG. 10 shows the carbohydrate profiles of a mutant and a transgenic low raffinose saccharide soybean compared to wild type soybean.

[0034] FIG. 11 shows vector pKR57.

[0035] FIG. 12 shows vector pKR63.

[0036] FIG. 13 shows vector pDS1.

[0037] FIG. 14 shows vector pKR72.

[0038] FIG. 15 shows vector pDS2.

[0039] FIG. 16 shows vector pDS3 (orientation 2).

[0040] FIG. 17 shows vector SH60.

[0041] FIG. 18 shows a TLC analysis of somatic embryos containing the beta-conglycinin/KTI3 driven recombinant expression construct described in Example 13.

[0042] SEQ ID NO:1 is the 1151 bp sequence derived from clone sdp3c.pk013.c9 (FIS) of the soybean nucleotide sequence containing the ORF [nucleotides 71-1090 (Stop)] of the galactinol synthase 3 gene.

[0043] SEQ ID NO:2 is the 339 amino acid sequence encoded by the ORF [nucleotides 71-1090 (Stop)] of SEQ ID NO: 1

[0044] SEQ ID NO:3 is the 1398 bp sequence derived from clone srr3c.pk003.h12 (FIS) of the soybean nucleotide sequence containing the ORF [nucleotides 94-1089 (Stop)] of the galactinol synthase 4 gene.

[0045] SEQ ID NO:4 is the 331 amino acid sequence encoded by the ORF [nucleotides 94-1089 (Stop)] of SEQ ID NO: 3.

[0046] SEQ ID NO:5 is the 1417 bp sequence derived from clone srr3c.pk001.i20 (FIS) of the soybean nucleotide sequence containing the ORF [nucleotides 213-1187 (Stop)] of the galactinol synthase 5 gene.

[0047] SEQ ID NO:6 is the 324 amino acid sequence encoded by the ORF [nucleotides 213-1187 (Stop)] of SEQ ID NO: 5

[0048] SEQ ID NO:7 is the amino acid sequence of the galactinol synthase from Pisum sativum (NCBI GenBank Identifier (GI) No. 5541885).

[0049] SEQ ID NO:8 is the amino acid sequence of the galactinol synthase from Arabidopsis thaliana (NCBI GenBank Identifier (GI) No. 15223567).

[0050] SEQ ID NO:9 is the amino acid sequence of the galactinol synthase from Glycine max (NCBI GenBank Identifier (GI) No. 32345694).

[0051] SEQ ID NO:10 represents the 1406 bp of the soybean nucleotide sequence of the galactinol synthase 1 gene.

[0052] SEQ ID NO:11 represents the 1350 bp of the soybean nucleotide sequence galactinol synthase 2 gene.

[0053] SEQ ID NO:12 is the forward primer used to amplify part of galactinol synthase 1 as described in Example 6.

[0054] SEQ ID NO:13 is the reverse primer used to amplify part of galactinol synthase 1 as described in Example 6.

[0055] SEQ ID NO:14 is the 519 bp sequence amplified from the galactinol synthase 1 gene (SEQ ID NO:10) as described in Example 6.

[0056] SEQ ID NO:15 is the forward primer used to amplify part of galactinol synthase 2 as described in Example 6.

[0057] SEQ ID NO:16 is the reverse primer used to amplify part of galactinol synthase 2 as described in Example 6.

[0058] SEQ ID NO:17 is the 519 bp sequence amplified from the galactinol synthase 2 gene (SEQ ID NO:11) as described in Example 6.

[0059] SEQ ID NO:18 is the forward primer used to amplify part of galactinol synthase 3 as described in Example 6.

[0060] SEQ ID NO:19 is the reverse primer used to amplify part of galactinol synthase 3 as described in Example 6.

[0061] SEQ ID NO:20 is the 519 bp sequence amplified from the galactinol synthase 3 gene (SEQ ID NO:1) as described in Example 6.

[0062] SEQ ID NO:21 is the forward primer used to isolate and amplify the soybean PM29 promoter as described in Example 10.

[0063] SEQ ID NO:22 is the reverse primer used to isolate and amplify the soybean PM29 promoter as described in Example 10.

[0064] SEQ ID NO:23 is the 597 bp sequence of the soybean PM29 promoter.

[0065] SEQ ID NO:24 is the forward primer used to re-amplify the PM29 promoter as described in Example 11.

[0066] SEQ ID NO:25 is the reverse primer used to re-amplify the PM29 promoter as described in Example 11.

[0067] SEQ ID NO:26 is the sequence of two copies of the Eag1-ELVISLIVES sequence as described in Example 11.

[0068] SEQ ID NO:27 represents the sequence of the complementary strand of SEQ ID NO: 26.

[0069] SEQ ID NO:28 represents the sequence of a truncated version of the two copies of the ELVISLIVES (ELEL) linker.

[0070] SEQ ID NO:29 is the 8810 bp sequence of vector SH50.

[0071] SEQ ID NO:30 is the 4479 bp sequence of vector pKR57.

[0072] SEQ ID NO:31 is the 5010 bp sequence of vector pKR63.

[0073] SEQ ID NO:32 is the 5414 bp sequence of v pDS1.

[0074] SEQ ID NO:33 is the 7085 bp sequence of vector pKR72.

[0075] SEQ ID NO:34 is the 5303 bp sequence of vector pDS2.

[0076] SEQ ID NO:35 is the 8031 bp sequence of vector pDS3 (orientation 2).

[0077] SEQ ID NO:36 is the 9616 bp sequence of vector SH60.

[0078] SEQ ID NO:37 is the 1585 bp sequence of the Not1 fragment of vector pJMS10 (FIG. 3) described in Example 13.

[0079] 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 37 C.F.R. .sctn.1.821-1.825.

[0080] 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.

DETAILED DESCRIPTION OF THE INVENTION

[0081] In the context of this disclosure, a number of terms shall be utilized. The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid fragment"/"isolated 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. An isolated polynucleotide of the present invention may include at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from SEQ ID NOs:1 or 3 or 5, or the complement of such sequences.

[0082] The term "isolated" refers to materials, such as nucleic acid molecules and/or proteins, 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.

[0083] 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.

[0084] A "recombinant DNA construct" comprises any of the isolated polynucleotides of the present invention operably linked to at least one regulatory sequence.

[0085] As used herein, "substantially similar" refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. "Substantially similar" also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. "Substantially similar" also refers 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 affect the functional properties of the resulting transcript vis-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms "substantially similar" and "corresponding substantially" are used interchangeably herein.

[0086] "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.

[0087] 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.

[0088] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp (1989) CABIOS. 5: 151-153) and found in the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). The "default parameters" are the parameters preset by the manufacturer of the program and for multiple alignments they correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10, while for pairwise alignments they are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. After alignment of the sequences, using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.

[0089] Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

[0090] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment, which result in the production of a chemically equivalent amino acid at a given site, but do not affect 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 which 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. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence of SEQ ID NOs:1, 3 or 5 and the complement of such nucleotide sequences may be used to affect the expression and/or function of a galactinol synthase in a host cell. A method of using an isolated polynucleotide to affect the level of expression of a polynucleotide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated recombinant DNA construct of the present invention; introducing the isolated polynucleotide or the isolated recombinant DNA construct into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain isolated polynucleotide.

[0091] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridization, IRL Press, Oxford, U.K.). 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 uses 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 uses 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 uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

[0092] 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 about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

[0093] 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%. 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.

[0094] A "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0095] "Codon degeneracy" refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting 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.

[0096] "Synthetic nucleic acid fragments" can be assembled 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.

[0097] "Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Recombinant DNA construct" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a recombinant DNA construct 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. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign-gene" refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, recombinant DNA constructs, or recombinant DNA constructs. A "transgene" is an isolated nucleic acid fragment or recombinant DNA construct that has been introduced into the genome by a transformation procedure.

[0098] "Coding sequence" refers to a nucleotide sequence that codes for a specific amino acid sequence. "Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0099] "Promoter" refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a nucleotide 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. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a 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, nucleic acid fragments of different lengths may have identical promoter activity.

[0100] "Convergent promoters" refers to promoters that are situated on either side of the isolated nucleic acid fragment of interest such that the direction of transcription from each promoter is opposing each other. Any promoter useful in plant transgene expression can be used. The promoters can be the same or different. The promoters are convergent with the isolated nucleic acid fragment being situated between the convergent promoters. It is important that the promoters have similar spatial and temporal activity, i.e., similar spatial and temporal patterns of expression, so that double-stranded RNA is produced in plants or plant organs by the recombinant construct that is stably integrated into the genome of the plant or plant organ. This has been described in U.S. provisional application 60/578,404, filed Jun. 9, 2004 which is filed simultaneously herewith. Also, this is described in U.S. provisional application 60/625,835, filed Nov. 8, 2004.

[0101] "Translation leader sequence" refers to a nucleotide sequence located between the promoter sequence 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 and Foster (1995) Mol. Biotechnol. 3:225-236).

[0102] "3' non-coding sequences" refer to nucleotide 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 et al. (1989) Plant Cell 1:671-680.

[0103] "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 posttranscriptional 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 polypeptides by the cell. "cDNA" refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. "Sense-RNA" refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. "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 (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0104] 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)).

[0105] 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).

[0106] 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.

[0107] 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 Al which was published on Dec. 30, 2004.

[0108] The term "operably linked" refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting 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 sense or antisense orientation.

[0109] The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to 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. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "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, incorporated herein by reference).

[0110] A "protein" or "polypeptide" is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.

[0111] "Altered levels" or "altered expression" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0112] "Mature protein" or the term "mature" when used in describing a 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" or the term "precursor" when used in describing a 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 but are not limited to intracellular localization signals.

[0113] "Transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or "gene gun" transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0114] "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. The term "transformation" as used herein refers to both stable transformation and transient transformation.

[0115] The terms "recombinant construct", "expression construct" and "recombinant expression construct" are used interchangeably herein. These terms refer to a functional unit of genetic material that can be inserted into the genome of a cell using standard methodology well known to one skilled in the art.

[0116] The term "vector" refers to a vehicle used for gene cloning to insert a foreign nucleic acid fragment into the genome of a host cell.

[0117] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis").

[0118] "Motifs" or "subsequences" refer to short regions of conserved sequences of nucleic acids or amino acids that comprise part of a longer sequence. For example, it is expected that such conserved subsequences would be important for function, and could be used to identify new homologues in plants. It is expected that some or all elements may be found in a homologue. Also, it is expected that one or two of the conserved amino acids in any given motif may differ in a true homologue.

[0119] "PCR" or "polymerase chain reaction" is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0120] The present invention concerns an isolated polynucleotide comprising:

[0121] (a) a nucleotide sequence encoding a polypeptide having galactinol synthase activity, wherein the polypeptide has an amino acid sequence of at least 85% identity, when compared to one of SEQ ID NO: 2 or 4 or 95% identity when compared to one of SEQ ID NO:6, based on the Clustal V method of alignment,

[0122] (b) all or part of the isolated polynucleotide comprising (a) for use in co-suppression or antisense suppression of endogenous nucleic acid sequences encoding polypeptides having galactinol synthase activity, or

[0123] (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.

[0124] This invention also includes the isolated complement of such polynucleotides, wherein the complement and the polynucleotide consist of the same number of nucleotides, and the nucleotide sequence of the complement and the polynucleotide have 100% complementarity.

[0125] Nucleic acid fragments encoding at least a portion of several galactinol synthases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0126] For example, genes encoding other galactinol synthases, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0127] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, or 5 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a galactinol synthase polypeptide.

[0128] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0129] In another embodiment, this invention concerns viruses and host cells comprising either the recombinant DNA constructs of the invention as described herein or isolated polynucleotides of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.

[0130] 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.

[0131] 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.

[0132] As was noted above, the nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of galactinol synthase, galactinol, and raffinose saccharides in those cells.

[0133] Overexpression of the proteins of the instant invention may be accomplished by first constructing a recombinant DNA construct in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The recombinant DNA construct may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided. The instant recombinant DNA construct may also comprise one or more introns in order to facilitate gene expression of the recombinant DNA construct.

[0134] Plasmid vectors comprising the instant isolated polynucleotides) (or recombinant DNA constructs) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the recombinant DNA construct or recombinant DNA construct. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0135] It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a recombinant DNA construct designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a recombinant DNA construct designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense recombinant DNA constructs could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

[0136] Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of a specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

[0137] The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different recombinant DNA constructs utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

[0138] In still another embodiment, the present invention concerns a galactinol synthase polypeptide having an amino acid sequence comprising at least 85% identical, based on the Clustal method of alignment, to a polypeptide of SEQ ID NO:2 or 4 or at least 95% identical to a polypeptide of SEQ ID NO:6. The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a recombinant DNA construct for production of the instant polypeptides. This recombinant DNA construct could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded galactinol synthase. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 5).

[0139] All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0140] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0141] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0142] Nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0143] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0144] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptide. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptide can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

[0145] This invention also relates to a method of reducing the raffinose saccharide content of soybean seeds by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% or any integer percentage in between 30% to 100%.

[0146] Raffinose saccharides are a group of D-galactose-containing oligosaccharide derivatives of sucrose that are widely distributed in plants. Raffinose saccharides are characterized by the following general formula: [O-.beta.-D-galactopyranosyl-(1.fwdarw.6).sub.n-.alpha.-glucopyr- anosyl-(1.fwdarw.2)-.beta.-D-fructofuranoside where n=0 through n=4 are known respectively as sucrose, raffinose, stachyose, verbascose and ajugose.

[0147] More specifically, this invention concerns method for reducing the level of at least one raffinose saccharide in soybean comprising: [0148] (a) constructing a recombinant DNA construct comprising all or part of at least one isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having galactinol synthase activity for use in co-suppression or antisense suppression of endogenous nucleic acid sequences encoding polypeptides having galactinol synthase activity operably linked to at least one regulatory sequence; and [0149] (b) transforming a soybean cell with the recombinant DNA construct of (a); and [0150] (c) regenerating soybean plants from the transformed cells of step (c); and

[0151] screening seeds obtained from the plants of (c) for an altered level of galactinol synthase in the transformed soybean cell when compared to a corresponding nontransformed soybean cell.

[0152] The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988)). 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. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. 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 is cultivated using methods well known to one skilled in the art.

[0153] 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.

[0154] Methods for transforming dicots, primarily using Agrobacterium tumefaciens, and obtaining transgenic plants have been published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al. (1988) Bio/Technology 6:923, Christou et al. (1988) Plant Physiol. 87:671-674); 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 and pea (Grant et al. (1995) Plant Cell Rep. 15:254-258).

[0155] Transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium have also been reported. Transformation and plant regeneration have been achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) (1987) 84:5354); barley (Wan and Lemaux (1994) Plant Physiol. 104:37); Zea mays (Rhodes et al. (1988) Science 240:204, Gordon-Kamm et al. (1990) Plant Cell 2:603-618, Fromm et al. (1990) Bio/Technology 8:833; Koziel et al. (1993) Bio/Technology 11: 194, Armstrong et al. (1995) Crop Science 35:550-557); oat (Somers et al. (1992) Bio/Technology 10: 15 89); orchard grass (Horn et al. (1988) Plant Cell Rep. 7:469); rice (Toriyama et al. (1986) Theor. Appl. Genet. 205:34; Part et al. (1996) Plant Mol. Biol. 32:1135-1148; Abedinia et al. (1997) Aust. J. Plant Physiol. 24:133-141; Zhang and Wu (1988) Theor. Appl. Genet. 76:835; Zhang et al. (1988) Plant Cell Rep. 7:379; Battraw and Hall (1992) Plant Sci. 86:191-202; Christou et al. (1991) Bio/Technology 9:957); rye (De la Pena et al. (1987) Nature 325:274); sugarcane (Bower and Birch (1992) Plant J. 2:409); tall fescue (Wang et al. (1992) Bio/Technology 10:691), and wheat (Vasil et al. (1992) Bio/Technology 10:667; U.S. Pat. No. 5,631,152).

[0156] Assays for gene expression based on the transient expression of cloned nucleic acid constructs have been developed by introducing the nucleic acid molecules into plant cells by polyethylene glycol treatment, electroporation, or particle bombardment (Marcotte et al., Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989); McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev. 6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

[0157] Transient expression systems may be used to functionally dissect isolated nucleic acid fragment constructs (see generally, Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995)). It is understood that any of the nucleic acid molecules of the present invention can be introduced into a plant cell in a permanent or transient manner in combination with other genetic elements such as vectors, promoters, enhancers etc.

[0158] In addition to the above discussed procedures 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 organisms and screening and isolating of clones (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989); Maliga et al., Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995); Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor, N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, Cold Spring Harbor, N.Y. (1998); Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer, New York (1997)) are well known.

EXAMPLES

[0159] The present invention is further defined 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.

[0160] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1

Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0161] A cDNA library representing mRNAs from soybean (Glycine max) tissue was prepared. The characteristics of the library are described below.

TABLE-US-00001 TABLE 1 cDNA Libraries from Soybean Library Tissue Clone sdp3c Soybean (Glycine max [L.]) sdp3c.pk013.c9 developing pods 8-9 mm srr3c Soybean (Glycine max [L.], Bell) srr3c.pk003.h12:fis roots control for src3c. srr3c.pk001.i20:fis

[0162] 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.

[0163] Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.

[0164] Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.

[0165] Sequence data is collected (ABI Prism Collections) and assembled using Phred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).

[0166] In some of the clones the cDNA fragment corresponds to a portion of the 3'-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information one of two different protocols are used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution. In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5'-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3'-untranslated region (also referred to as UTR). In the second round of amplification a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above.

Example 2

Identification of cDNA Clones

[0167] cDNA clones encoding galactinol synthase 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.

[0168] ESTs submitted for analysis are compared to the GenBank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLAST algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLAST algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.

Example 3

Characterization of cDNA Clones Encoding Galactinol Synthase

[0169] The BLASTX search using the EST sequences from the clones listed in Table 2 revealed similarity of the polypeptides encoded by the cDNAs to galactinol synthase from Pisum sativum (NCBI GenBank Identifier (GI) No. 5541885, SEQ ID NO:7), Arabidopsis thaliana (NCBI GenBank Identifier (GI) No. 15223567, SEQ ID NO:8) and Glycine max (NCBI GenBank Identifier (GI) No. 32345694, SEQ ID NO:9). Shown in Table 2 are the BLAST results for the sequences encoding an entire protein ("CGS") derived from the entire cDNA inserts comprising the indicated cDNA clones ("fis"):

TABLE-US-00002 TABLE 2 LAST Results for Sequences Encoding Polypeptides Homologous to Galactinol Synthase BLAST pLog Score Clone Status (NCBI) sdp3c.pk013.c9:fis CGS 149.57 (GI:5541885) (SEQ ID NO:2) srr3c.pk003.h12:fis CGS 135.89 (GI:15223567) (SEQ ID NO:4) srr3c.pk001.i20:fis CGS 166.70 (GI:32345694) (SEQ ID NO:6)

[0170] The sequence of the entire cDNA insert in the clones listed in Table 2 was determined. The data in Table 3 represent a calculation of the percent identity of the amino acid sequences set forth in SEQ ID Nos: 2, 4, and 6 and the sequences of Pisum sativum (SEQ ID NO: 7), Arabidopsis thaliana (SEQ ID NO: 8) and Glycine max (SEQ ID NO: 9).

TABLE-US-00003 TABLE 3 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Galactinol Synthase Percent Identity to Clone SEQ ID NO: (Accession No.) sdp3c.pk013.c9:fis 2 82 (GI:5541885) srr3c.pk003.h12:fis 4 75 (GI:15223567) srr3c.pk001.i20:fis 6 92 (GI:32345694)

[0171] 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 and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a galactinol synthase. These sequences represent new soybean sequences encoding galactinol synthase.

[0172] The expression pattern of galactinol synthase 3, 4 and 5 during soybean seed development was analyzed via Lynx MPSS Brenner et al (2000) Proc Natl Acad Sci USA 97:1665-70) and is shown in Table 4.

TABLE-US-00004 TABLE 4* 15 20 30 40 45 50 55 Clone Designation DAF DAF DAF DAF DAF DAF DAF mature sdp3c.pk013.c9 -- -- -- 6 143 796 1979 1604 srr3c.pk003.h12 -- -- -- -- 21 151 152 133 srr3c.pk001.i20 -- -- 13 -- 45 79 365 329 *Lynx MPSS profiles (expressed as adjusted PPM) of galactinol synthase 3 (sdp3c.pk013.c9), galactinol synthase 4 (srr3c.pk003.h12) and galactinol synthase 5 (srr3c.pk001.i20) during soybean seed development (DAF = days after flowering, mature = mature seed).

[0173] The results shown in Table 4 demonstrate that expression of all three galactinol synthases are only detectable during the later stages of seed development (45 DAF to mature seed). Galactinol synthase 4 is very lowly expressed compared to galactinol synthase 3 and 5, which show high and intermediate expression levels during late seed development. The pattern of expression between galactinol synthase 3 and 5 also differs: whereas galactinol synthase 3 expression levels increase during the course of late seed development, reaching a plateau in the mature seed, galactinol synthase 5 expression appears to be prominent mainly at 55 DAF and in mature seed.

Example 4

Expression of Recombinant DNA Constructs in Dicot Cells

[0174] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the alpha 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 cassette 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 cassette is flanked by Hind III sites.

[0175] 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 expression cassette.

[0176] 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.

[0177] Soybean embryogenic suspension cultures can be maintained in 35 mL 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.

[0178] 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 PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0179] A selectable marker gene which can be used to facilitate soybean transformation is a recombinant DNA construct 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 expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptide 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.

[0180] 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.

[0181] 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 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.

[0182] 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 5

Expression of Recombinant DNA Constructs in Microbial Cells

[0183] 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.

[0184] 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% low melting agarose gel. 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 (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 .quadrature.L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB), 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 polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0185] 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.

[0186] Crude, partially purified or purified enzyme, either alone or as a fusion protein, may be utilized in assays for the evaluation of enzymatic activity of the instant polypeptides disclosed herein. Assays may be conducted under well-known experimental conditions which permit optimal enzymatic activity. Assays for galactinol synthase activity are presented by Odegard and Lumen (1995) Plant Physiol. 109: 505-511.

Example 6

Construction of Chimeric Vectors for Seed-Targeted Co-Suppression of Galactinol Synthase in Transgenic Glycine max

[0187] Vectors designed for the seed-specific co-suppression of galactinol synthase 1 galactinol synthase 2 and galactinol synthase 3 in soybean were assembled as described below.

Amplification of Partial Galactinol Synthase Polynucleotides

[0188] Polynucleotide fragments encoding parts of the galactinol synthase 1 (GAS1 (SEQ ID NO:6 of U.S. Pat. Nos. 5,773,699 and 5,648,210), galactinol synthase 2 (GAS2) in clone ses4d.pk0017.b8 (WO 01/77306) and galactinol synthase 3 (GAS3) in clone sdp3c.pk013.c9 were amplified by standard PCR methods using Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.) and the following primer sets. The GAS1 oligonucleotide primers were designed to add a Not I restriction endonuclease site at the Send and a XhoI site to the 3' end (SEQ ID NO:12 and SEQ ID NO:13, respectively). The DNA sequence comprising the 519 bp polynucleotide from soybean GAS1 is shown in SEQ ID NO:14.

[0189] The GAS2 oligonucleotide primers were designed to add a XhoI restriction endonuclease site at the Send and a PstI site to the 3' end (SEQ ID NO:15 and SEQ ID NO:16 respectively). The DNA sequence comprising the 519 bp polynucleotide from soybean GAS2 is shown in SEQ ID NO:17.

[0190] The GAS3 oligonucleotide primers were designed to add a PstI restriction endonuclease site at the Send and a NotI site to the 3' end (SEQ ID NO:18 and 19, respectively). The DNA sequence comprising the 519 bp polynucleotide from soybean GAS3 is shown in SEQ ID NO:20.

Assembly of Vectors for the Co-Suppression of Galactinol Synthase

[0191] Preparation of pJMS08: The polynucleotide products for GAS1, GAS2 and GAS3 obtained from the amplifications described above were digested with Not I, Xho1 and PSt1 and assembled into vector pJMS08 (FIG. 2) by the following steps. First, the plasmid KS151 [US patent publication 2003/0036197A1] was digested with Not I. Then, the isolated DNA fragments containing partial sequences of GAS1, GAS2 and GAS3 were inserted into Not I-digested plasmid KS151 to obtain plasmid pJMS08 (FIG. 2).

[0192] Plasmid KS151 also comprises nucleotides encoding HPT under the control of the T7 promoter and termination signals and the 35S promoter and Nos 3' terminator (US patent publication 2003/0036197A1). The KTi3 promoter and 3' transcription terminator region have been described by Jofuku et al. ((1989) Plant Cell 1:1079-1093). The KTI3 promoter directs strong embryo-specific expression of transgenes.

[0193] Preparation of pJMS10: The polynucleotide products for GAS1, GAS2 and GAS3 obtained from the amplifications described above were digested with Not I, Xho1 and PSt1 and assembled into vector pJMS10 (FIG. 3) by the following steps. From plasmid KS123 (prepared according to US Application No. 2004/0073975 A1, which published on Apr. 15, 2004) the HindIII cassette containing the beta-conglycinin promoter-phaseolin terminator was removed creating the plasmid KS120. To the unique BamHI site of plasmid KS120 a lea promoter-phaseolin terminator was inserted as a BamHI fragment creating plasmid KS127. The Lea promoter (Lee et al (1992) Plant Physiol. 100:2121-2122; Genbank Accession no. M97285) was amplified from genomic A2872 soybean DNA and a phaseolin 3' end was added as described in US patent publication 2003/0036197 A1. To KS127 an EL linker was added to a unique Not1 site as described in US patent publication 2003/0036197 A1, creating plasmid KS139. To KS139 an EL linker was added to a unique Not1 site as described in US patent publication 2003/0036197 A1, creating plasmid KS147. Plasmid KS147 also comprises nucleotides encoding HPT under the control of the T7 promoter and termination signals and the 35S promoter and Nos 3'. Then, the isolated DNA fragments containing partial sequences of GAS1, GAS2 and GAS3 were inserted into the Not I-digested plasmid KS147 to obtain plasmid pJMS10 (FIG. 3).

Example 7

Construction of Chimeric Vectors for Seed-Targeted Co-Suppression of Galactinol Synthase in Transgenic Glycine max

[0194] Vectors designed for the seed-specific co-suppression of galactinol synthase 3, galactinol synthase 4 and galactinol synthase 5 in soybean can be assembled as described below.

Amplification of Partial Galactinol Synthase Polynucleotides

[0195] Polynucleotide fragments encoding parts of the galactinol synthase 4 (SEQ ID NO: 3), galactinol synthase 5 (SEQ ID NO:5) and galactinol synthase 3 (SEQ ID NO:1) are amplified by standard PCR methods using Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.). Appropriate primer sets are chosen, giving polynucleotide fragments of similar length as described for GAS1, 2 and 3 (Example 6), which is well within the routine skill in the art.

Assembly of Vectors for the Co-Suppression of Galactinol Synthase

[0196] The assembly of vectors containing GAS 3, 4, and 5 for the co-suppression of galactinol synthase is performed essentially as described for GAS 1, 2 and 3 in Example 6.

[0197] Transformation into soybean somatic embryos and carbohydrate analysis will be performed as described below for GAS 1, 2, and 3.

[0198] It is expected, that a similar reduction in Raffinose Saccharides in soybean seeds will be observed using GAS 3, 4, 5 as the one observed with GAS1, 2, and 3.

Example 8

Transformation of Soybean Somatic Embryos with Galactinol Synthase Co-Suppression Vectors

[0199] To study the possibility of reducing Raffinose Family Oligosaccharides (RFOs), soybean somatic embryos were transformed with the seed-specific expression vectors pJMS08 (FIG. 2) or pJMS10 (FIG. 3) by the method of particle gun bombardment (Klein, T. M. et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050). Soybean somatic embryos from the Jack cultivar were induced as follows. Cotyledons (3 mm in length) were dissected from surface sterilized, immature seeds and were cultured for an additional 6-10 weeks in the light at 26.degree. C. on a Murashige and Skoog media containing 7 g/L agar and supplemented with 10 mg/mL 2,4-D. Globular stage somatic embryos, which produced secondary embryos, were then excised and placed into flasks containing liquid MS medium supplemented with 2,4-D (10 mg/mL) and cultured in the light on a rotary shaker. After repeated selection for clusters of somatic embryos that multiplied as early, globular staged embryos, the soybean embryogenic suspension cultures were maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with fluorescent lights on a 16:8 hour day/night schedule. Cultures were subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0200] Soybean embryogenic suspension cultures were then transformed by the method of particle gun bombardment using a DuPont Biolistic.TM. PDS1000/HE instrument (helium retrofit). To 50 .mu.L of a 60 mg/.mu.L 1 mm gold particle suspension were added (in order): 5 .mu.L of 1 mg/.mu.L DNA (pJMS01 plus pJMS02, pRM02 plus pRM03, pRM01, or pRM04), 20 .mu.L of 0.1 M spermidine, and 50 .mu.L of 2.5 M CaCl.sub.2. The particle preparation was then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles were then washed once in 400 .mu.L 70% ethanol and resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle suspension was sonicated three times for one second each. Five .mu.L of the DNA-coated gold particles was then loaded on each macro carrier disk.

[0201] Approximately 300-400 mg of a two-week-old suspension culture was 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 to 10 plates of tissue were bombarded. Membrane rupture pressure was set at 1100 psi and the chamber was evacuated to a vacuum of 28 inches mercury. The tissue was placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue was divided in half and placed back into liquid and cultured as described above.

[0202] Five to seven days post bombardment, the liquid media was exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media was refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue was observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue was removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line was treated as an independent transformation event. These suspensions were then subcultured and maintained as clusters of immature embryos.

[0203] These immature soybean embryos were dried-down (by transferring them into an empty small petridish that was seated on top of a 10 cm petridish containing some agar gel to allow slow dry down) to mimic the last stages of soybean seed development.

[0204] Dried-down embryos are capable of producing plants when transferred to soil or soil-less media. Storage products produced by embryos at this stage are similar in composition to storage products produced by zygotic embryos at a similar stage of development and most importantly the storage product profile is predictive of plants derived from a somatic embryo line (WO 94/11516, published May 26, 1994)).

Example 9

Carbohydrate Analysis of Transgenic Soybean Somatic Embryos

[0205] The carbohydrate composition of transgenic somatic embryos identified in Example 6 as containing the pJMS08 or pJMS10 cassettes was measured by high performance anion exchange chromatography/pulsed amperometric detection (HPAE/PAD). Fresh individual somatic embryos from transgenic lines were rapidly washed in water, dried on a paper towel, and transferred into 1.5 mL microcentrifuge tubes. Ethanol (80%) was added to the tubes and the samples were heated to 70.degree. C. for 15 minutes. The samples were centrifuged at 14,000 rpm for 5 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.

[0206] For HPAE analysis, 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 25 minute linear gradient of 0.5 to 170 mM NaAc in 150 mM NaOH at a flow rate of 1.0 mL/min. Soluble sugars were identified by comparison to standards (glucose, fructose, sucrose, raffinose, stachyose, and verbascose) using HPAE/PAD.

[0207] FIG. 4 and Table 5 show a typical carbohydrate profile resulting from HPAE/PAD analysis of transgenic soybean somatic embryos co-suppressing galactinol synthase. A clear reduction in RFOs (raffinose, stachyose and vebascose) can be observed as compared to FIG. 5 and wild type values in Table 5.

[0208] FIG. 5 shows a typical carbohydrate profile resulting from HPAE/PAD analysis of a soybean somatic embryo showing a wild type carbohydrate phenotype.

[0209] The results for two different events showing cosuppression experiments of the three isoforms of Galactinol Synthases 1, 2, and 3 are shown in Table 5 above. For each event, 6 seeds were analyzed. The results are expressed in .mu.mol/g dwt (sugar unit), where the dry weight calculation was based on 7% moisture content of seed.

TABLE-US-00005 TABLE 5 Stach/ Phen % Event seed wt(g) gol sucr raff stach verb totRSA raff o-type reduction 1231- 1 0.14 1.37 179.99 18.35 11.90 0.14 43.94 0.65 Low 48.45 1-1-1 RFO 1231- 2 0.14 1.12 120.71 19.84 32.33 0.00 85.62 1.63 WT 1-1-1 1231- 3 0.19 0.68 159.38 18.06 20.57 0.00 59.88 1.14 Low 29.77 1-1-1 RFO 1231- 4 0.20 0.83 122.36 17.84 31.62 0.99 84.88 1.77 WT 1-1-1 1231- 5 0.19 0.78 148.20 16.14 19.20 0.00 55.32 1.19 Low 35.10 1-1-1 RFO 1231- 6 0.14 0.57 161.80 15.75 12.98 0.00 42.28 0.82 Low 50.41 1-1-1 RFO Mean 0.17 0.97 121.49 18.84 31.97 0.49 85.24 1.70 0.00 WT Mean 0.16 0.85 162.34 17.07 16.16 0.03 50.35 0.95 40.93 Low RFO 1231- 1 0.13 2.12 187.39 21.03 13.56 0.41 51.51 0.64 Low 52.67 1-1-3 RFO 1231- 2 0.13 2.15 186.36 25.69 16.09 0.43 61.30 0.63 Low 43.68 1-1-3 RFO 1231- 3 0.13 2.09 193.18 25.31 33.42 0.00 94.25 1.32 WT 1-1-3 1231- 4 0.15 0.81 231.88 20.46 8.90 0.21 39.72 0.44 Low 63.51 1-1-3 RFO 1231- 5 0.14 1.28 236.19 19.82 13.43 0.19 48.54 0.68 Low 55.40 1-1-3 RFO 1231- 6 0.15 1.63 164.68 17.05 43.41 1.11 108.84 2.55 WT 1-1-3 Mean 0.14 1.86 178.93 21.18 38.41 0.56 101.55 0.00 WT Mean 0.14 1.59 210.46 21.75 12.98 0.31 50.27 50.50 Low RFO

[0210] TotRSA (total raffinose saccharides) refers to the .alpha.-galactose content present in the sum of galactinol (gol, 1 mol .alpha.-galactose/mole), raffinose (raft, 1 mol .alpha.-galactose/mole), stachyose (stach, 2 mol .alpha.-galactose/mole) and verbascose (verb, 3 mol .alpha.-galactose/mole). Sucrose is sucr. % Reduction indicates the change in total RFOs compared to the wild type.

Example 10

Isolation of Soybean PM29 Promoter

[0211] The promoter of a soybean seed maturation protein was isolated using a polymerase chain reaction (PCR) based approach. Soybean genomic DNA was digested to completion with a DNA restriction enzyme that generates blunt ends (DraI, EcoRV, PvuII or StuI, for example) according to standard protocols. The Universal GenomeWalker.TM. kit from Clonetech.TM. (Product User Manual No. PT3042-1) was used to ligate adaptors to the ends of the genomic DNA fragments. Nested primers are also supplied in the Universal GenomeWalker.TM. kit that are specific for the adaptor sequence (AP1 and AP2, for the first and second adaptor primer, respectively). Two gene specific primers (GSP1 and GSP2) were designed for the soybean PM29 gene based on the 5' coding sequences in PM29 cDNA in DuPont EST database. The oligonucleotide sequences of the GSP1 and GSP2 primers (SEQ ID NO:21 and SEQ ID NO:22, respectively) contain recognition sites for the restriction enzyme BAMH I.

[0212] The AP2 primer from the Universal GenomeWalker.TM. kit contains a Sal I restriction site. The AP1 and the GSP1 primers were used in the first round PCR using each of the adaptor ligated genomic DNA populations (DraI, EcoRV, PvuII or StuI) under conditions defined in the GenomeWalker.TM. protocol. Cycle conditions were 94.degree. C. for 4 minutes; 94.degree. C. for 2 seconds and 72.degree. C. for 3 minutes, 7 cycles; 94.degree. C. for 2 seconds and 67.degree. C. for 3 minutes, 32 cycles; 67.degree. C. for 4 minutes. The products from each of the first run PCRs were diluted 50-fold. One microliter from each of the diluted products was used as templates for the second PCR with the AP2 and GSP2 as primers. Cycle conditions were 94.degree. C. for 4 minutes; 94.degree. C. for 2 seconds and 72.degree. C. for 3 minutes, 5 cycles; 94.degree. C. for 2 seconds and 67.degree. C. for 3 minutes, 20 cycles; 67.degree. C. for 3 minutes. Agarose gels were run to determine which PCR gave an optimal fragment length. A 679 bp genomic fragment was detected and isolated from the EcoRV-digested genomic DNA reaction. The genomic fragment was digested with BamH I and Sal I and cloned into Bluescript KS.sup.+ vector for sequencing. Finally, sequencing data indicated that this genomic fragment contained a 597 bp soybean PM29 promoter sequence as shown in SEQ ID NO:23.

Example 11

Construction of Galactinol Synthase Silencing Plasmids Driven by PM29

[0213] Two oligonucleotides were designed to re-amplify the PM29 promoter with either BamH I or Nco I sites (SEQ ID NO:24 and SEQ ID NO:25, respectively). The re-amplified PM29 promoter fragment was digested with BamH I and Nco I, purified and cloned into the BamH I and Nco I sites of plasmid pG4G (FIG. 6) to make the fusion between the soybean PM29 promoter-GUS fusion (pSH43). The plasmid pG4G has been described in U.S. No. 5,968,793, the contents of which are hereby incorporated by reference.

Preparation of SH55 and SH49:

[0214] Plasmid pSH43 (described above) was digested with NcoI, filled in with vent polymerase (obtained from New England Biolabs Inc.) and subsequently digested with BamHI (5' end of the promoter). The resulting promoter fragment was isolated and cloned into pBluescript II SK (+) (Stratagene, Inc.) previously cut with XbaI (and filled in by vent polymerase) and BamHI creating the plasmids pBluescriptPM29. This construct contains a unique Not1 site at the 3' end of the promoter. Two copies of the Eag1-ELVISLIVES sequence (SEQ ID NO:26) were added on the 5' site of the Not1 site as described in EP1297163 A2 (PCT Publication No. WO 2002/000904, which published Jan. 3, 2002).

[0215] The promoter fragment was isolated using a BamHI/Not1 digestion and ligated into pJMS10 plasmid previously cut with BamH1 (partial) and Not1. The pJMS10 plasmid also contains the complementary strand of SEQ ID NO:26 (SEQ ID NO:27) 3' of the Not1 site. This ligation resulted in the following plasmid: SH55 (PM29 promoter-ELEL-Not1-ELEL-Phaseolin terminator) and SH49. SH49 is identical to SH55 with the exception of a truncated ELEL sequence (SEQ ID NO:28) at the 3' border of the Not1. The truncated sequence is missing the "tgacca" of the ELEL sequence at the 3' border of the Not1 and was identified after sequence verification of the plasmid and probably originated during the PCR amplification of the ELEL linker. This truncation has no effect on the ability of silencing the GAS genes as is evident from Example 12.

Preparation of SH50:

[0216] A Not1 fragment containing the partial sequences of soybean GAS1 (SEQ ID NO:14), GAS2 (SEQ ID NO:17) and GAS3 (SEQ ID NO:20) was digested from pJMS10 (described above) and then ligated into SH49 previously digested with Not1, creating the plasmid SH50 (SEQ ID NO:29 and FIG. 7).

Example 12

Raffinose Family Oligosaccharide (RFO) Analysis of PM29 Driven Transgenic Soybean Somatic Embryos and Mature Seeds

[0217] Soybean somatic embryos were transformed with a seed-preferred expression vector SH50 (SEQ ID NO:29 and FIG. 7) by the method described in Example 8.

[0218] Raffinose Family Oligosaccharides (galactinol, raffinose, stachyose, etc.) of transgenic somatic embryos and seeds containing the PM29 promoter driven recombinant expression construct described in Example 11 was measured by thin layer chromatography. Somatic embryos or seed chips were extracted with hexane then dried. The dried material was resuspended in 80% methanol, incubated at room temperature for 1-2 hours, centrifuged, and 2 microliters of the supernatant is spotted onto a TLC plate (Kieselgel 60 CF, from EM Scientific, Gibbstown, N.J.; Catalog No. 13749-6). The TLC was run in ethylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5 hours. The air dried plates were sprayed with 2% sulfuric acid and heated until the charred sugars were detected. As shown in FIG. 8 two lines show reduced levels of raffinose sugars (raffinose and stachyose lowest bands) when compared to a to wild-type soybean. The arrow indicates somatic embryos with reduced raffinose family oligosaccharides. (WT=wild type control, S=sucrose, Rf=raffinose and St=stachyose standard). FIG. 9 shows a TLC analysis of mature seed chips from a soybean line transformed with SH50 and revealed an almost complete reduction in raffinose and stachyose (RFO) sugars in seeds when compared to wild-type soybean. The plate shows that 13 out of 17 seeds from a single event show a dramatic reduction in raffinose family oligosaccharides (RFO).

Carbohydrate Analysis of Transgenic Soybean Seeds

[0219] The carbohydrate composition of transgenic soybean seeds containing the PM29 promoter driven recombinant expression construct described in Example 11 (SH50) was measured by high performance anion exchange chromatography/pulsed amperometric detection (HPAE/PAD). Seed chips were extracted in ethanol (80%) and heated to 70.degree. C. for 15 minutes. The samples were centrifuged at 14,000 rpm for 5 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.

[0220] For HPAE analysis, 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 20 isocratic run in 150 mM NaOH at a flow rate of 1.0 mL/min. Soluble sugars were identified by comparison to standards (glucose, fructose, sucrose, raffinose, stachyose, and verbascose) using HPAE/PAD.

[0221] A typical profile of a soybean mutant characterized by highly reduced raffinose family oligosaccharides content (Hitz et al. 2002. Plant Physiology Vol 128, pp 650-660) is shown in FIG. 10 (Mutant HE2, left). As a comparison, the carbohydrate profile of soybeans transformed with SH50 as described in Example 12 is shown on the right (Transgenic seed with HE2 phenotype). Furthermore, the sucrose to RFO ratio of the transgenic and mutant was very similar and more than 10 fold higher when compared to wild type. The % decrease in RFO when compared to wild type of some transgenic seeds are shown in Table 6 and indicate a % reduction ranging from 71% to 89%. In comparison, the percent reduction of the mutant was 85%.

TABLE-US-00006 TABLE 6 Sucrose to RFO ratio and % decrease in RFO of transgenic soybean seeds (transformed with SH50) when compared with Jack wild type. A U V 1 Event S/RFO ratio % decrease in RFO 2 AFS4042-5-1-1, seed 1 6.8 71 3 AFS4042-5-1-1, seed 5 15.9 87 4 AFS4042-5-1-1, seed 7 11.2 82 5 AFS4042-5-1-1, seed 12 12.7 85 6 AFS4042-5-1-1, seed 18 17.8 89 7 AFS4042-5-1-1, seed 20 12.5 82 8 AFS4042-5-1-3, seed 7 16.3 87 9 AFS4042-5-1-4, seed 2 14.6 85 10 AFS4042-5-1-4, seed 7 10.5 80 11 12 Jack-wild type 0 14 Low RFO mutant 85 The % reduction in RFO of a known mutant is included as a reference.

Example 13

Construction of Galactinol Synthase Silencing Plasmids Driven by .beta.-Conglycinin and KTI3

[0222] Plasmid pJMS10 (FIG. 3) was prepared as described in Example 6.

[0223] Preparation of Plasmid pDS3:

[0224] pKR57 (FIG. 11) (4479 bp; SEQ ID NO:30) was digested with Eco RI and Not I, run on a 0.8% Tris-Acetate-Ethylenediaminetetraacetic acid-agarose gel (TAE-agarose gel) and a 3144 bp fragment containing the .beta.-conglycinin promoter, an origin of replication and a gene encoding ampicillin resistance was purified using the Qiagen gel extraction kit. pKR63 (FIG. 12) (5010 bp; SEQ ID NO:31) was digested with Eco RI and Not I, run on a 0.8% TAE-agarose gel and a 2270 bp fragment containing the KTi promoter was purified using the Qiagen gel extraction kit. The isolated fragments were ligated together and the ligation was transformed into E. coli and colonies were selected on ampicillin. Bacterial colonies were selected and grown overnight in LB media and appropriate antibiotic selection. DNA was isolated from the resulting culture using a Qiagen miniprep kit according to the manufacturer's protocol and then analyzed by restriction digest. The resulting plasmid was named pDS1 (FIG. 13) (5414 bp; SEQ ID NO:32).

[0225] pKR72 (FIG. 14) (7085 bp; SEQ ID NO:33) was digested with Hind III, run on a 0.8% TAE-agarose gel and a 5303 bp fragment containing a gene that encodes resistance to hygromycin operably linked to a prokaryotic promoter and a gene that encodes resistance to hygromycin operably linked to a eukaryotic promoter were purified using the Qiagen gel extraction kit. The fragment was ligated to itself and the ligation was transformed into E. coli and colonies were selected on hygromycin. Bacterial colonies were selected and grown overnight in LB media and appropriate antibiotic selection. DNA was isolated from the resulting culture using a Qiagen miniprep kit according to the manufacturer's protocol and then analyzed by restriction digest. The resulting plasmid was named pDS2 (FIG. 15) (5303 bp; SEQ ID NO:34).

[0226] pDS2 was digested with Sal I and the ends were dephosphorylated with calf intestinal alkaline phosphatase (CIAP) according to the manufacture's instructions (Stratagene, San Diego, Calif.). pDS1 was digested with Sal I and Fsp I, run on a 0.8% TAE-agarose gel and a 2728 bp fragment containing the KTi3 promoter and the .beta.-conglycinin promoter in opposite orientations was purified using the Qiagen gel extraction kit. The isolated fragments were ligated together and the ligation was transformed into E. coli and colonies were selected on hygromycin. Bacterial colonies were selected and grown overnight in LB media and appropriate antibiotic selection. DNA was isolated from the resulting culture using a Qiagen miniprep kit according to the manufacturer's protocol and then analyzed by restriction digest. The resulting plasmids were named pDS3 [orientation 2 (FIG. 16, SEQ ID NO: 35)].

[0227] Preparation of SH60:

[0228] pJMS10 (FIG. 3) was digested with Not1, run on a 0.8% TAE-agarose gel and a 1585 bp DNA fragment (SEQ ID NO:37) comprising the partial sequences of GAS1 (SEQ ID NO:14), GAS2 (SEQ ID NO:17) and GAS3 (SEQ ID NO:20) was purified using the Qiagen gel extraction kit. pDS3 (orientation 2) (FIG. 16, SEQ ID NO: 35) was digested with Not1, run on a 0.8% TAE-agarose gel and a 8031 bp DNA fragment was purified using the Qiagen gel extraction kit. The isolated fragments were ligated together and the ligation was transformed into E. coli and colonies were selected on hygromycin. Bacterial colonies were selected and grown overnight in LB media and appropriate antibiotic selection. DNA was isolated from the resulting culture using a Qiagen miniprep kit according to the manufacturer's protocol and then analyzed by restriction digest. The resulting plasmid was named SH60 (FIG. 17, SEQ ID NO: 36).

Example 14

Reduction of Raffinose Family Oligosaccharide (RFO) in Transgenic Soybean Somatic Embryos

[0229] SH60, as described in Example 13, was transformed into soybean embryogenic suspension cultures using a protocol as described in Example 8 above. Individual immature soybean embryos were dried-down (by transferring them into an empty small petridish that was seated on top of a 10 cm petridish containing some agar gel to allow slow dry down) to mimic the last stages of soybean seed development. Dried-down embryos are capable of producing plants when transferred to soil or soil-less media. Storage products produced by embryos at this stage are similar in composition to storage products produced by zygotic embryos at a similar stage of development and most importantly the storage product profile is predictive of plants derived from a somatic embryo line (PCT Publication No. WO 94/11516, which published on May 26, 1994). Raffinose Family Oligosaccharides (raffinose, stachyose) of transgenic somatic embryos containing the B-conglycinin/KTI3 driven (SH60) recombinant expression construct described in Example 13 was measured by thin layer chromatography. Somatic embryos were extracted with hexane then dried. The dried material was re-suspended in 80% methanol, incubated at room temperature for 1-2 hours, centrifuged, and 2 .mu.l of the supernatant is spotted onto a TLC plate (Kieselgel 60 CF, from EM Scientific, Gibbstown, N.J.; Catalog No. 13749-6). The TLC was run methylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5 hours. The air dried plates were sprayed with 2% sulfuric acid and heated until the charred sugars were detected. As shown in FIG. 18 the embryos labeled "Low RFO embryos" show reduced levels of raffinose sugars (raffinose and stachyoses) when compared to a to wild-type soybean. Five out of eleven (45%) lines analyzed showed reduced levels of RFOs, which is demonstrative of reduced galactinol synthase expression (see Table 7).

TABLE-US-00007 TABLE 7 Positive Transformed Lines with Reduced Galactinol Synthase Expression carbohydrate phenotype GAS1GAS2GAS3 lines with wild type 6 out of 11 RFO levels GAS1GAS2GAS3 lines with reduced 5 out of 11 RFO levels Percent gene silencing 45%

Sequence CWU 1

1

3711151DNAGlycine maxCDS(71)..(1090) 1ctaagctctc ttttagtctt actcacaaac acttttttca ctgcttccat tacgaacata 60tatttattat atg gct cct gaa ctt gtc ccc acc gtt gtg aaa tcc agt 109 Met Ala Pro Glu Leu Val Pro Thr Val Val Lys Ser Ser 1 5 10gct gcg ttc acg aaa ccc gcg acc ctt cca agg cgt gcc tac gtg aca 157Ala Ala Phe Thr Lys Pro Ala Thr Leu Pro Arg Arg Ala Tyr Val Thr 15 20 25ttc ctc gcc gga aac ggt gac tac gtg aaa ggg gtg gtt ggc ctc gcc 205Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu Ala30 35 40 45aaa ggg ttg cga aag gtg aaa acc gcg tac ccg ttg gtg gtg gct gtc 253Lys Gly Leu Arg Lys Val Lys Thr Ala Tyr Pro Leu Val Val Ala Val 50 55 60ctc ccc gat gtg ccg gag gag cac cgt aag atc ctg gag tct cag ggc 301Leu Pro Asp Val Pro Glu Glu His Arg Lys Ile Leu Glu Ser Gln Gly 65 70 75tgc atc gtt cgc gag atc gaa ccc gtt tac cca ccc gaa aac caa acc 349Cys Ile Val Arg Glu Ile Glu Pro Val Tyr Pro Pro Glu Asn Gln Thr 80 85 90cag ttt gcc atg gct tat tac gtc atc aac tac tcc aag ctc cgt ata 397Gln Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile 95 100 105tgg gag ttt gtg gag tac agc aag atg ata tac ttg gac gga gac att 445Trp Glu Phe Val Glu Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp Ile110 115 120 125gag gta tat gag aac ata gac cac cta ttt gac cta cct gat ggt aac 493Glu Val Tyr Glu Asn Ile Asp His Leu Phe Asp Leu Pro Asp Gly Asn 130 135 140ttt tac gct gtg atg gat tgt ttc tgc gag aag aca tgg agt cac acc 541Phe Tyr Ala Val Met Asp Cys Phe Cys Glu Lys Thr Trp Ser His Thr 145 150 155cct cag tac aag gtg ggt tac tgc cag caa tgc ccg gag aag gtg cgg 589Pro Gln Tyr Lys Val Gly Tyr Cys Gln Gln Cys Pro Glu Lys Val Arg 160 165 170tgg ccc acc gaa ttg ggt cag ccc cct tct ctt tac ttc aac gct ggc 637Trp Pro Thr Glu Leu Gly Gln Pro Pro Ser Leu Tyr Phe Asn Ala Gly 175 180 185atg ttc gtg ttc gaa ccc aac atc gcc acc tat cat gac cta ttg aaa 685Met Phe Val Phe Glu Pro Asn Ile Ala Thr Tyr His Asp Leu Leu Lys190 195 200 205acg gtg caa gtc acc act ccc acc tcg ttc gct gaa caa gat ttc ttg 733Thr Val Gln Val Thr Thr Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu 210 215 220aac atg tac ttc aag gac att tac aag cca atc cct tta aat tac aat 781Asn Met Tyr Phe Lys Asp Ile Tyr Lys Pro Ile Pro Leu Asn Tyr Asn 225 230 235ctt gtc ctc gcc atg ctg tgg cgc cac ccg gaa aac gtt aaa tta gac 829Leu Val Leu Ala Met Leu Trp Arg His Pro Glu Asn Val Lys Leu Asp 240 245 250caa gtc aag gtt gtt cac tat tgc gca gcg ggg tcc aag cca tgg aga 877Gln Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg 255 260 265tat acg ggg aag gaa gag aat atg cag agg gag gac ata aag atg ctg 925Tyr Thr Gly Lys Glu Glu Asn Met Gln Arg Glu Asp Ile Lys Met Leu270 275 280 285gtg aag aaa tgg tgg gat atc tac aat gat gct tcg ctt gac tac aag 973Val Lys Lys Trp Trp Asp Ile Tyr Asn Asp Ala Ser Leu Asp Tyr Lys 290 295 300cca ttg atg aat gca agt gaa gct cca gca gcg gat ggt gtt gac att 1021Pro Leu Met Asn Ala Ser Glu Ala Pro Ala Ala Asp Gly Val Asp Ile 305 310 315gaa caa ttc gtg cag gct cta tca gag gtt ggt cat gtt caa tat gtc 1069Glu Gln Phe Val Gln Ala Leu Ser Glu Val Gly His Val Gln Tyr Val 320 325 330acc gcg cct tca gca gct taa ttaagagggc acattcaaat cacgacaaaa 1120Thr Ala Pro Ser Ala Ala 335aacaaccaag tgaaaaaaaa aaaaaaaaaa a 11512339PRTGlycine max 2Met Ala Pro Glu Leu Val Pro Thr Val Val Lys Ser Ser Ala Ala Phe1 5 10 15Thr Lys Pro Ala Thr Leu Pro Arg Arg Ala Tyr Val Thr Phe Leu Ala 20 25 30Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu 35 40 45Arg Lys Val Lys Thr Ala Tyr Pro Leu Val Val Ala Val Leu Pro Asp 50 55 60Val Pro Glu Glu His Arg Lys Ile Leu Glu Ser Gln Gly Cys Ile Val65 70 75 80Arg Glu Ile Glu Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala 85 90 95Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe 100 105 110Val Glu Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp Ile Glu Val Tyr 115 120 125Glu Asn Ile Asp His Leu Phe Asp Leu Pro Asp Gly Asn Phe Tyr Ala 130 135 140Val Met Asp Cys Phe Cys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr145 150 155 160Lys Val Gly Tyr Cys Gln Gln Cys Pro Glu Lys Val Arg Trp Pro Thr 165 170 175Glu Leu Gly Gln Pro Pro Ser Leu Tyr Phe Asn Ala Gly Met Phe Val 180 185 190Phe Glu Pro Asn Ile Ala Thr Tyr His Asp Leu Leu Lys Thr Val Gln 195 200 205Val Thr Thr Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr 210 215 220Phe Lys Asp Ile Tyr Lys Pro Ile Pro Leu Asn Tyr Asn Leu Val Leu225 230 235 240Ala Met Leu Trp Arg His Pro Glu Asn Val Lys Leu Asp Gln Val Lys 245 250 255Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr Thr Gly 260 265 270Lys Glu Glu Asn Met Gln Arg Glu Asp Ile Lys Met Leu Val Lys Lys 275 280 285Trp Trp Asp Ile Tyr Asn Asp Ala Ser Leu Asp Tyr Lys Pro Leu Met 290 295 300Asn Ala Ser Glu Ala Pro Ala Ala Asp Gly Val Asp Ile Glu Gln Phe305 310 315 320Val Gln Ala Leu Ser Glu Val Gly His Val Gln Tyr Val Thr Ala Pro 325 330 335Ser Ala Ala31398DNAGlycine maxCDS(94)..(1089) 3gcacgaggtg atttttgctt aattactaaa ccaaaccatt tcttattccc tcatcgaaac 60cttttctttc tatatatttc ccttttcaat atc atg gca cct aac atc acc acc 114 Met Ala Pro Asn Ile Thr Thr 1 5gtt gtt gcc aat gcc acc act gag caa tta ccc aaa gct cat gga gga 162Val Val Ala Asn Ala Thr Thr Glu Gln Leu Pro Lys Ala His Gly Gly 10 15 20agt agt ggg cgt gcc ttt gtg act ttt ctt gct gga aac ggt gat tat 210Ser Ser Gly Arg Ala Phe Val Thr Phe Leu Ala Gly Asn Gly Asp Tyr 25 30 35gta aag ggt gtt gtg ggt ttg gcc aaa gga ctg aga aag gcc aaa agc 258Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu Arg Lys Ala Lys Ser40 45 50 55atg tac cct ttg gtg gtt gct gtg tta cca gat gtt cct gaa gaa cat 306Met Tyr Pro Leu Val Val Ala Val Leu Pro Asp Val Pro Glu Glu His 60 65 70cgt gcg att ctc aaa tcc caa ggt tgc att gtc agg gag att gaa cct 354Arg Ala Ile Leu Lys Ser Gln Gly Cys Ile Val Arg Glu Ile Glu Pro 75 80 85gtg tac cct cct aag aac cag acc cag ttc gcc atg gcc tat tat gtc 402Val Tyr Pro Pro Lys Asn Gln Thr Gln Phe Ala Met Ala Tyr Tyr Val 90 95 100atc aat tac tcc aag cta cgt att tgg gag ttc gtg gag tac cag aag 450Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Tyr Gln Lys 105 110 115atg ata tac cta gac ggc gac atc caa gtt ttt gga aac att gac cac 498Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe Gly Asn Ile Asp His120 125 130 135ttg ttt gat ctt cct aat aat tat ttc tat gcg gtg atg gat tgt ttc 546Leu Phe Asp Leu Pro Asn Asn Tyr Phe Tyr Ala Val Met Asp Cys Phe 140 145 150tgc gag aag act tgg agc cac acc cct cag ttc cag att ggg tac tgc 594Cys Glu Lys Thr Trp Ser His Thr Pro Gln Phe Gln Ile Gly Tyr Cys 155 160 165caa cag tgc cct gat aag gtt caa tgg ccc tct cac ttt ggt acc aaa 642Gln Gln Cys Pro Asp Lys Val Gln Trp Pro Ser His Phe Gly Thr Lys 170 175 180cct cct cta tat ttc aat gct ggc atg ttt gtt tat gag cct aat ctc 690Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val Tyr Glu Pro Asn Leu 185 190 195aac acc tac cgt cat ctt ctc caa act gtc caa gtc atc aag ccc acg 738Asn Thr Tyr Arg His Leu Leu Gln Thr Val Gln Val Ile Lys Pro Thr200 205 210 215tcc ttt gct gag cag gac ttt ctg aac atg tac ttc aag gac aag tac 786Ser Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Lys Tyr 220 225 230aag cca ata ccg aac gtg tac aac ctt gtg ctg gcc atg ttg tgg cgt 834Lys Pro Ile Pro Asn Val Tyr Asn Leu Val Leu Ala Met Leu Trp Arg 235 240 245cac cct gag aat gtt gaa ctt gat caa gtt caa gtg gtt cat tac tgt 882His Pro Glu Asn Val Glu Leu Asp Gln Val Gln Val Val His Tyr Cys 250 255 260gct gct ggg tct aag cct tgg agg ttc act ggg aag gaa gag aac atg 930Ala Ala Gly Ser Lys Pro Trp Arg Phe Thr Gly Lys Glu Glu Asn Met 265 270 275gat agg gaa gat atc aag atg ctt atg aag aag tgg tgg gac ata tat 978Asp Arg Glu Asp Ile Lys Met Leu Met Lys Lys Trp Trp Asp Ile Tyr280 285 290 295gaa gat gag aca ctg gac tac aat aac aac tct gtc aat gtg gaa cgt 1026Glu Asp Glu Thr Leu Asp Tyr Asn Asn Asn Ser Val Asn Val Glu Arg 300 305 310ttc aca tca gta cta ttg gat gct ggg ggt ttt cag ttt gtg cca gca 1074Phe Thr Ser Val Leu Leu Asp Ala Gly Gly Phe Gln Phe Val Pro Ala 315 320 325cct tct gct gcc taa tatgattcac agctacaaat taaagtctaa ttaacgacaa 1129Pro Ser Ala Ala 330agtatatatg tattgttatt tgtttttgtt ttttttttcg tttttgggtc ttatgaacga 1189accacgtcta tagttttaat ttggatgacc tttttgtata caaagtcaca tgtgacgtct 1249tacagctttt gattattatt aagatttaat tatatgagtc ctttacttaa tttgttttca 1309ttgatcaaga gttgtggata tatatatata tatatatata tctttaattt tattaaatga 1369aattttaagg caaaaaaaaa aaaaaaaaa 13984331PRTGlycine max 4Met Ala Pro Asn Ile Thr Thr Val Val Ala Asn Ala Thr Thr Glu Gln1 5 10 15Leu Pro Lys Ala His Gly Gly Ser Ser Gly Arg Ala Phe Val Thr Phe 20 25 30Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu Ala Lys 35 40 45Gly Leu Arg Lys Ala Lys Ser Met Tyr Pro Leu Val Val Ala Val Leu 50 55 60Pro Asp Val Pro Glu Glu His Arg Ala Ile Leu Lys Ser Gln Gly Cys65 70 75 80Ile Val Arg Glu Ile Glu Pro Val Tyr Pro Pro Lys Asn Gln Thr Gln 85 90 95Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp 100 105 110Glu Phe Val Glu Tyr Gln Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln 115 120 125Val Phe Gly Asn Ile Asp His Leu Phe Asp Leu Pro Asn Asn Tyr Phe 130 135 140Tyr Ala Val Met Asp Cys Phe Cys Glu Lys Thr Trp Ser His Thr Pro145 150 155 160Gln Phe Gln Ile Gly Tyr Cys Gln Gln Cys Pro Asp Lys Val Gln Trp 165 170 175Pro Ser His Phe Gly Thr Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met 180 185 190Phe Val Tyr Glu Pro Asn Leu Asn Thr Tyr Arg His Leu Leu Gln Thr 195 200 205Val Gln Val Ile Lys Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn 210 215 220Met Tyr Phe Lys Asp Lys Tyr Lys Pro Ile Pro Asn Val Tyr Asn Leu225 230 235 240Val Leu Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Gln 245 250 255Val Gln Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg Phe 260 265 270Thr Gly Lys Glu Glu Asn Met Asp Arg Glu Asp Ile Lys Met Leu Met 275 280 285Lys Lys Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu Asp Tyr Asn Asn 290 295 300Asn Ser Val Asn Val Glu Arg Phe Thr Ser Val Leu Leu Asp Ala Gly305 310 315 320Gly Phe Gln Phe Val Pro Ala Pro Ser Ala Ala 325 33051417DNAGlycine maxCDS(213)..(1184) 5ccaagatctt aaaatatctc ttccatacaa gtttgttttc aaagtgtttt tgtctcccaa 60atcctactct tgtgaccaca agccttcact tcactctctc tctctctctc tctctctctc 120tctctctctc tctctctttt ttgaaaccct tttttctctt ctcaaaccaa accaagcaag 180caattatatt acactactca ctcactgaga cc atg gct cct aat atc acc acc 233 Met Ala Pro Asn Ile Thr Thr 1 5gtc acc gac gct caa gcc aag gcc gcc ggc ggg cgt ggc cgt gcc tac 281Val Thr Asp Ala Gln Ala Lys Ala Ala Gly Gly Arg Gly Arg Ala Tyr 10 15 20gtc acc ttc ctc gcc gga aac ggt gac tat gtg aaa ggt gtc gtt ggc 329Val Thr Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly 25 30 35ttg gcc aaa ggt ctg agg aag gtg aaa agc atg tac cct ctg gtg gtt 377Leu Ala Lys Gly Leu Arg Lys Val Lys Ser Met Tyr Pro Leu Val Val40 45 50 55gca gtg tta ccc gat gtt cca gaa cat cac cga aac att ctc acc tcc 425Ala Val Leu Pro Asp Val Pro Glu His His Arg Asn Ile Leu Thr Ser 60 65 70caa ggt tgc att gtt aga gaa att gaa ccc gtg tac cct cct gag aat 473Gln Gly Cys Ile Val Arg Glu Ile Glu Pro Val Tyr Pro Pro Glu Asn 75 80 85cag acg cag ttc gcc atg gca tat tac gtc atc aac tat tcc aag cta 521Gln Thr Gln Phe Ala Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu 90 95 100cgt att tgg gag ttt gtg gag ttc agc aag atg ata tac cta gac ggt 569Arg Ile Trp Glu Phe Val Glu Phe Ser Lys Met Ile Tyr Leu Asp Gly 105 110 115gat ata caa gtg ttt gac aat att gac cac ttg ttt gac ttg cct gat 617Asp Ile Gln Val Phe Asp Asn Ile Asp His Leu Phe Asp Leu Pro Asp120 125 130 135aac tac ttt tat gcg gtg atg gac tgt ttt tgt gag ccc act tgg ggc 665Asn Tyr Phe Tyr Ala Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly 140 145 150cac act ctg cag tat caa atc gga tac tgc cag cag tgc cct cat aag 713His Thr Leu Gln Tyr Gln Ile Gly Tyr Cys Gln Gln Cys Pro His Lys 155 160 165gtt cag tgg ccc act cac ttt ggg ccc aag cct cct ctc tat ttc aat 761Val Gln Trp Pro Thr His Phe Gly Pro Lys Pro Pro Leu Tyr Phe Asn 170 175 180gct ggc atg ttt gtt tat gag ccc aat ctg gat acc tac cgt gac ctc 809Ala Gly Met Phe Val Tyr Glu Pro Asn Leu Asp Thr Tyr Arg Asp Leu 185 190 195ctt caa act gtc caa gtc act aag ccc act tcc ttt gct gaa cag gat 857Leu Gln Thr Val Gln Val Thr Lys Pro Thr Ser Phe Ala Glu Gln Asp200 205 210 215ttt ttg aac atg tac ttc aag gac aaa tat agg cca att cct aat gtc 905Phe Leu Asn Met Tyr Phe Lys Asp Lys Tyr Arg Pro Ile Pro Asn Val 220 225 230tat aat ctt gtg ttg gcc atg ctg tgg cgt cac cct gag aac gtt gag 953Tyr Asn Leu Val Leu Ala Met Leu Trp Arg His Pro Glu Asn Val Glu 235 240 245ctt gaa aaa gtt aaa gtg gtt cac tac tgt gct gct gga tct aag cct 1001Leu Glu Lys Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro 250 255 260tgg agg tac aca ggg aag gag gaa aat atg gag aga gaa gat atc aag 1049Trp Arg Tyr Thr Gly Lys Glu Glu Asn Met Glu Arg Glu Asp Ile Lys 265 270 275atg ttg gtg aag aag tgg tgg gat ata tat gag gat gag act ttg gac 1097Met Leu Val Lys Lys Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu Asp280 285 290 295tac aac aat cca ttc aac gtg gat agg ttc act gcg gca ctt ttg gag 1145Tyr Asn Asn Pro Phe Asn Val Asp Arg Phe Thr Ala Ala Leu Leu Glu 300 305 310gtt ggt gaa gtc aag ttc gtc cgt gcc cca tct gct gct tagagtgtct 1194Val Gly Glu Val Lys Phe Val Arg Ala Pro Ser Ala Ala 315 320ttggaaatca agtgtgatcc aagtacatag gataagatat acagacccat acatcattaa 1254gttttatgtg tttttaaagt gtttagagga cctttttatg tgtccctttt ttcttttttc 1314tttttcaatt ctgccattgt aaagcagtga aataccatgt ccttaatttt attattggat 1374atgaatttta ttttgtacgt

tctctaaaaa aaaaaaaaaa aaa 14176324PRTGlycine max 6Met Ala Pro Asn Ile Thr Thr Val Thr Asp Ala Gln Ala Lys Ala Ala1 5 10 15Gly Gly Arg Gly Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 20 25 30Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu Arg Lys Val Lys 35 40 45Ser Met Tyr Pro Leu Val Val Ala Val Leu Pro Asp Val Pro Glu His 50 55 60His Arg Asn Ile Leu Thr Ser Gln Gly Cys Ile Val Arg Glu Ile Glu65 70 75 80Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala Met Ala Tyr Tyr 85 90 95Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Phe Ser 100 105 110Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe Asp Asn Ile Asp 115 120 125His Leu Phe Asp Leu Pro Asp Asn Tyr Phe Tyr Ala Val Met Asp Cys 130 135 140Phe Cys Glu Pro Thr Trp Gly His Thr Leu Gln Tyr Gln Ile Gly Tyr145 150 155 160Cys Gln Gln Cys Pro His Lys Val Gln Trp Pro Thr His Phe Gly Pro 165 170 175Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val Tyr Glu Pro Asn 180 185 190Leu Asp Thr Tyr Arg Asp Leu Leu Gln Thr Val Gln Val Thr Lys Pro 195 200 205Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Lys 210 215 220Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu Ala Met Leu Trp225 230 235 240Arg His Pro Glu Asn Val Glu Leu Glu Lys Val Lys Val Val His Tyr 245 250 255Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn 260 265 270Met Glu Arg Glu Asp Ile Lys Met Leu Val Lys Lys Trp Trp Asp Ile 275 280 285Tyr Glu Asp Glu Thr Leu Asp Tyr Asn Asn Pro Phe Asn Val Asp Arg 290 295 300Phe Thr Ala Ala Leu Leu Glu Val Gly Glu Val Lys Phe Val Arg Ala305 310 315 320Pro Ser Ala Ala7334PRTPisum sativum 7Met Ala Pro Glu Ile Val Gln Thr Ser Thr Lys Pro Val Thr Gly Phe1 5 10 15Thr Lys Leu Lys Arg Ala Tyr Val Thr Phe Leu Ala Gly Asn Gly Asp 20 25 30Tyr Val Lys Gly Val Ile Gly Leu Ala Lys Gly Leu Arg Lys Val Lys 35 40 45Thr Ala Tyr Pro Leu Val Val Ala Val Leu Pro Asp Val Pro Glu Glu 50 55 60His Arg Glu Met Leu Glu Ser Gln Gly Cys Ile Val Arg Glu Ile Gln65 70 75 80Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala Met Ala Tyr Tyr 85 90 95Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Tyr Ser 100 105 110Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Tyr Glu Asn Ile Asp 115 120 125His Leu Phe Asp Leu Pro Asp Gly Tyr Phe Tyr Ala Val Met Asp Cys 130 135 140Phe Cys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr Lys Ile Gly Tyr145 150 155 160Cys Gln Gln Cys Pro Glu Lys Val Gln Trp Pro Lys Glu Met Gly Glu 165 170 175Pro Pro Ser Leu Tyr Phe Asn Ala Gly Met Phe Leu Phe Glu Pro Ser 180 185 190Val Glu Thr Tyr Asp Asp Leu Leu Lys Thr Cys Gln Val Thr Ala Pro 195 200 205Thr Pro Phe Ala Asp Gln Asp Phe Leu Asn Met Tyr Phe Lys Asp Ile 210 215 220Tyr Arg Pro Ile Pro Leu Val Tyr Asn Leu Val Leu Ala Met Leu Trp225 230 235 240Arg His Pro Glu Asn Val Glu Leu Arg Lys Val Lys Val Val His Tyr 245 250 255Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr Thr Gly Lys Glu Glu Asn 260 265 270Met Gln Arg Glu Asp Ile Lys Met Leu Val Gln Lys Trp Leu Asp Ile 275 280 285Tyr Ser Asp Ser Ser Leu Asp Tyr Lys Lys Asn Leu Ser Gly Asn Cys 290 295 300Glu Thr Gln Arg Asn Asp Val Glu Glu Pro Phe Val Gln Ala Leu Ser305 310 315 320Glu Val Gly Arg Val Arg Tyr Val Thr Ala Pro Ser Ala Ala 325 3308335PRTArabidopsis thaliana 8Met Ala Pro Glu Ile Asn Thr Lys Leu Thr Val Pro Val His Ser Ala1 5 10 15Thr Gly Gly Glu Lys Arg Ala Tyr Val Thr Phe Leu Ala Gly Thr Gly 20 25 30Asp Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu Arg Lys Ala 35 40 45Lys Ser Lys Tyr Pro Leu Val Val Ala Val Leu Pro Asp Val Pro Glu 50 55 60Asp His Arg Lys Gln Leu Val Asp Gln Gly Cys Val Val Lys Glu Ile65 70 75 80Glu Pro Val Tyr Pro Pro Glu Asn Gln Thr Glu Phe Ala Met Ala Tyr 85 90 95Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe Val Glu Tyr 100 105 110Asn Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe Asp Asn Ile 115 120 125Asp His Leu Phe Asp Leu Pro Asn Gly Gln Phe Tyr Ala Val Met Asp 130 135 140Cys Phe Cys Glu Lys Thr Trp Ser His Ser Pro Gln Tyr Lys Ile Gly145 150 155 160Tyr Cys Gln Gln Cys Pro Asp Lys Val Thr Trp Pro Glu Ala Lys Leu 165 170 175Gly Pro Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val Tyr Glu 180 185 190Pro Asn Leu Ser Thr Tyr His Asn Leu Leu Glu Thr Val Lys Ile Val 195 200 205Pro Pro Thr Leu Phe Ala Glu Gln Asp Phe Leu Asn Met Tyr Phe Lys 210 215 220Asp Ile Tyr Lys Pro Ile Pro Pro Val Tyr Asn Leu Val Leu Ala Met225 230 235 240Leu Trp Arg His Pro Glu Asn Ile Glu Leu Asp Gln Val Lys Val Val 245 250 255His Tyr Cys Ala Ala Gly Ala Lys Pro Trp Arg Phe Thr Gly Glu Glu 260 265 270Glu Asn Met Asp Arg Glu Asp Ile Lys Met Leu Val Lys Lys Trp Trp 275 280 285Asp Ile Tyr Asn Asp Glu Ser Leu Asp Tyr Lys Asn Val Val Ile Gly 290 295 300Asp Ser His Lys Lys Gln Gln Thr Leu Gln Gln Phe Ile Glu Ala Leu305 310 315 320Ser Glu Ala Gly Ala Leu Gln Tyr Val Lys Ala Pro Ser Ala Ala 325 330 3359328PRTGlycine max 9Met Ala Pro Asn Ile Thr Thr Val Lys Thr Thr Ile Thr Asp Ala Gln1 5 10 15Ala Lys Val Ala Thr Asp His Gly Arg Ala Tyr Val Thr Phe Leu Ala 20 25 30Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu Ala Lys Gly Leu 35 40 45Arg Lys Val Lys Ser Met Tyr Pro Leu Val Val Ala Val Leu Pro Asp 50 55 60Val Pro Gln Asp His Arg Asn Ile Leu Thr Ser Gln Gly Cys Ile Val65 70 75 80Arg Glu Ile Glu Pro Val Tyr Pro Pro Glu Asn Gln Thr Gln Phe Ala 85 90 95Met Ala Tyr Tyr Val Ile Asn Tyr Ser Lys Leu Arg Ile Trp Glu Phe 100 105 110Val Glu Tyr Ser Lys Met Ile Tyr Leu Asp Gly Asp Ile Gln Val Phe 115 120 125Asp Asn Ile Asp His Leu Phe Asp Leu Pro Asp Asn Tyr Phe Tyr Ala 130 135 140Val Met Asp Cys Phe Cys Glu Pro Thr Trp Gly His Thr Lys Gln Tyr145 150 155 160Gln Ile Gly Tyr Cys Gln Gln Cys Pro His Lys Val Gln Trp Pro Thr 165 170 175His Phe Gly Pro Lys Pro Pro Leu Tyr Phe Asn Ala Gly Met Phe Val 180 185 190Tyr Glu Pro Asn Leu Ala Thr Tyr Arg Asp Leu Leu Gln Thr Val Gln 195 200 205Val Thr Gln Pro Thr Ser Phe Ala Glu Gln Asp Phe Leu Asn Ile Tyr 210 215 220Phe Lys Asp Lys Tyr Arg Pro Ile Pro Asn Val Tyr Asn Leu Val Leu225 230 235 240Ala Met Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Lys Val Lys 245 250 255Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg Tyr Thr Gly 260 265 270Lys Glu Glu Asn Met Glu Arg Glu Asp Ile Lys Met Leu Val Lys Lys 275 280 285Trp Trp Asp Ile Tyr Glu Asp Glu Thr Leu Asp Tyr Asn Asn Pro Leu 290 295 300Asn Val Asp Lys Phe Thr Ala Ala Leu Met Glu Val Gly Glu Val Lys305 310 315 320Phe Val Arg Ala Pro Ser Ala Ala 325101406DNAGlycine max 10gtttgttttc aaagtgtgtt ttgtttccca aatcctactc ttgtgaccac aacccttcct 60cctctttctt ttgaaacctc tttttttcta ttccccaacc aaacaagcaa acgctactca 120ctcatcatca ctgagatcat ggctcctaat atcaccactg tcaaaaccac catcaccgac 180gctcaagcca aggtcgccac cgatcatggt cgtgcctacg tcaccttcct cgccggaaac 240ggtgactatg tgaaaggtgt cgttggcttg gcaaaaggtc tgagaaaagt gaagagcatg 300taccctctgg tggttgcagt gctacccgat gttccccaag atcaccgcaa cattctcacc 360tcccaaggtt gcattgttag agagattgag cccgtgtacc ccccagagaa tcaaacccag 420tttgccatgg catattacgt catcaactat tccaagctac gtatttggga gtttgtggag 480tacagcaaga tgatatacct agacggtgat atccaagttt ttgacaacat tgaccacttg 540tttgacttgc ctgataacta cttctatgcg gtgatggact gtttctgtga gccaacttgg 600ggccacacta aacaatatca gatcggttac tgccagcagt gcccccataa ggttcagtgg 660cccactcact ttgggcccaa acctcctctc tatttcaatg ctggcatgtt tgtgtatgag 720cccaatttgg ctacttaccg tgacctcctt caaacagtcc aagtcaccca gcccacttcc 780tttgctgaac aggatttttt gaacatgtac ttcaaggaca aatataggcc aattcctaat 840gtctacaatc ttgtgctggc catgctgtgg cgtcaccctg agaacgttga gcttgacaaa 900gttaaagtgg ttcactactg tgctgctggg tctaagcctt ggaggtacac tgggaaggag 960gagaatatgg agagagaaga tatcaagatg ttagtgaaaa agtggtggga tatatatgag 1020gatgagactt tggactacaa caatccactc aatgtggata agttcactgc ggcacttatg 1080gaggttggtg aagtcaagtt cgtccgtgcc ccatctgctg cttaagagtg tctttggaaa 1140tcaagtgtga tccaagtaca tgtacaaagt catacatcat tacattaact tttatgtatt 1200tctaaaagtc atacatcatt acattaagtt ttatgtattt ctaaagtctt aagacttaag 1260aggacctttt ttatkkkkcc cgcttttctt tttttctttt tccaattctg tcattgtaaa 1320gsrgagaata ccgtatcctt aattttataa atggatatga attttatttg tactaaaggg 1380ggggccggta ccaattcgcc tatagt 1406111350DNAGlycine max 11gcacgagaaa caaccaacct cttcagtgat ctttgattag tactaagcta aaccatttct 60tattccctca aaatcaaaac ctttttcttt ctagctattt cccttttcaa atcatgccac 120ctaacatcac caccgttgtt gccaatgtca ccaccgagca attacccaag gctcgtggag 180gaagtgggcg tgccttcgtg acctttcttg ctgggaacgg tgattacgta aagggtgtcg 240tgggtttggc caaaggactg agaaaggcca aaagcatgta ccctttggtg gttgctgtgt 300taccagatgt tcctgaagaa catcgtgaga ttctcaaatc ccaaggttgc attgtcaggg 360agattgaacc tgtgtaccct cctgagaacc agacccagtt cgccatggcc tattatgtca 420tcaattactc caagctacgt atttgggagt tcgtggagta caagaagacg atatacctag 480acggtgacat ccaagtattt ggaaacatag accacttgtt tgatctgcct gataattatt 540tctatgcggt gatggattgt ttctgcgaga agacttggag ccacacccct cagttccaga 600ttgggtactg ccaacagtgc cctgataagg ttcaatggcc ctctcacttt ggttccaaac 660ctcctctata tttcaatgct ggcatgtttg tttatgagcc taatctcgac acctaccgtg 720atcttctcca aactgtccaa ctcaccaagc ccacttcttt tgctgagcag gactttctca 780acatgtactt caaggacaag tacaagccaa taccgaacat gtacaacctt gtgctggcca 840tgttgtggcg tcaccctgaa aatgttgaac ttgataaagt tcaagtggtt cattactgtg 900ctgctgggtc taagccttgg aggttcactg ggaaggaaga gaacatggat agggaagata 960tcaagatgct tgtgaagaag tggtgggaca tatatgaaga tgagacactg gactacaata 1020acaactctgt caacgtggaa cgtttcacat cggcactatt ggatgctggg ggctttcagt 1080ttgtgccagc accttctgct gcctaatatg cttattattt acagctacaa attaatgtta 1140attaacgaca aagtatatgt attgttattt gctttttttc gtttttgggt cttatatatg 1200aaggaacaac gtctatggtt ttaatttgga tgaccttctt gtatacaaag ccacatgtga 1260tctcatacag cttttgatta ttattaagaa attagaggac cttttattat gagtccttta 1320cttaaaaaaa aaaaaaaaaa aaaaaaaaaa 13501236DNAArtificialPrimer 12aagcttgcgg ccgcgtcatc aactattcca agctac 361336DNAArtificialPrimer 13aagcttctcg agtcacttcc cagtgtacct ccaagg 3614519DNAGlycine Max 14gtcatcaact attccaagct acgtatttgg gagtttgtgg agtacagcaa gatgatatac 60ctagacggtg atatccaagt ttttgacaac attgaccact tgtttgactt gcctgataac 120tacttctatg cggtgatgga ctgtttctgt gagccaactt ggggccacac taaacaatat 180cagatcggtt actgccagca gtgcccccat aaggttcagt ggcccactca ctttgggccc 240aaacctcctc tctatttcaa tgctggcatg tttgtgtatg agcccaattt ggctacttac 300cgtgacctcc ttcaaacagt ccaagtcacc cagcccactt cctttgctga acaggatttt 360ttgaacatgt acttcaagga caaatatagg ccaattccta atgtctacaa tcttgtgctg 420gccatgctgt ggcgtcaccc tgagaacgtt gagcttgaca aagttaaagt ggttcactac 480tgtgctgctg ggtctaagcc ttggaggtac actgggaag 5191534DNAArtificialPrimer 15aagcttctcg aggtcatcaa ttactccaag ctac 341643DNAArtificialPrimer 16agcttgcggc cgcctgcagt tacttcccag tgaacctcca agg 4317519DNAGlycine max 17gtcatcaatt actccaagct acgtatttgg gagttcgtgg agtacaagaa gacgatatac 60ctagacggtg acatccaagt atttggaaac atagaccact tgtttgatct gcctgataat 120tatttctatg cggtgatgga ttgtttctgc gagaagactt ggagccacac ccctcagttc 180cagattgggt actgccaaca gtgccctgat aaggttcaat ggccctctca ctttggttcc 240aaacctcctc tatatttcaa tgctggcatg tttgtttatg agcctaatct cgacacctac 300cgtgatcttc tccaaactgt ccaactcacc aagcccactt cttttgctga gcaggacttt 360ctcaacatgt acttcaagga caagtacaag ccaataccga acatgtacaa ccttgtgctg 420gccatgttgt ggcgtcaccc tgaaaatgtt gaacttgata aagttcaagt ggttcattac 480tgtgctgctg ggtctaagcc ttggaggttc actgggaag 5191842DNAArtificialPrimer 18aagcttgcgg ccgcctgcag gtcatcaact actccaagct cc 421937DNAArtificialPrimer 19aagcttgcgg ccgctacttc cccgtatatc tccatgg 3720519DNAGlycine max 20gtcatcaact actccaagct ccgtatatgg gagtttgtgg agtacagcaa gatgatatac 60ttggacggag acattgaggt atatgagaac atagaccacc tatttgacct acctgatggt 120aacttttacg ctgtgatgga ttgtttctgc gagaagacat ggagtcacac ccctcagtac 180aaggtgggtt actgccagca atgcccggag aaggtgcggt ggcccaccga attgggtcag 240cccccttctc tttacttcaa cgctggcatg ttcgtgttcg aacccaacat cgccacctat 300catgacctat tgaaaacggt gcaagtcacc actcccacct cgttcgctga acaagatttc 360ttgaacatgt acttcaagga catttacaag ccaatccctt taaattacaa tcttgtcctc 420gccatgctgt ggcgccaccc ggaaaacgtt aaattagacc aagtcaaggt tgttcactat 480tgcgcagcgg ggtccaagcc atggagatat acggggaag 5192126DNAArtificialPrimer 21tcttctgttc ttgccgttgc tttctc 262233DNAArtificialPrimer 22cgcggatccg acttgctcct tggcagcact ggt 3323597DNAGlycine max 23agagttttta taagttattt tatacatgaa ttaattttaa cttgtgaaaa aaattatttt 60cttcttataa gtatttatga caaagcttat ataaacatag tcttaatttc actcagaaaa 120acagaggagg aaaacttgtt gtatgaagcc cggctatttc atccattatc catatttgga 180tcgaaaagag aaggaaagtg tcattttata tgtgtataaa aagtatttca tccataagta 240atgataagat aattgtgtat gtaacattat taatgtattt aaattaaaat cataaattat 300tttaaacaat tcttattcgt tagtgacacg ataacggata agctaataat atatctatgg 360ttttctgtga acgtggcagc atattgatgg gaatagctct gcatgttgaa caagtggcac 420ggtacctagc gtgccttgct cttcttttgt ctaggcttgg tttggttcgc atcttccttc 480tcatataaat cctccaccac gtcgagtttt ctgttcaaat taaatcgttc aacactggaa 540ctctttgata taatatagaa agagacagag agagagagac agacaagaag aacaagg 5972442DNAArtificialPrimer 24cgcggatcca gagtttttat aagttatttt atacatgaat ta 422539DNAArtificialPrimer 25ccttgaccat ggttgttctt cttgtctgtc tctctctct 392681DNAArtificialtwo copies of Eag1-ELVISLIVES 26cggccggagc tggtcatctc gctcatcgtc gagtcggcgg ccggagctgg tcatctcgct 60catcgtcgag tcggcggccg c 812781DNAArtificialComplementary strand of two copies of Eag1-ELVISLIVES 27gcggccgccg actcgacgat gagcgagatg accagctccg gccgccgact cgacgatgag 60cgagatgacc agctccggcc g 812869DNAArtificialtruncated version of the two copies of ELVISLIVES linker 28gcggccgccg actcgacgat gagcgagatg accagctccg gccgccgact cgacgatgag 60cgagagctc 69298810DNAArtificialPlasmid SH50 29ggccgccgac tcgacgatga gcgagatgac cagctccggc cgccgactcg acgatgagcg 60agagctccgg ccgcaagtat gaactaaaat gcacgtaggt gtaagagctc atggagagca 120tggaatattg tatccgacca tgtaacagta taataactga gctccatctc acttcttcta 180tgaataaaca aaggatgtta tgatatatta acactctatc tatgcacctt attgttctat 240gataaatttc ctcttattat tataaatcat ctgaatcgtg acggcttatg gaatgcttca 300aatagtacaa aaacaaatgt gtactataag actttctaaa caattctaac tttagcattg 360tgaacgagac ataagtgtta agaagacata acaattataa tggaagaagt ttgtctccat 420ttatatatta

tatattaccc acttatgtat tatattagga tgttaaggag acataacaat 480tataaagaga gaagtttgta tccatttata tattatatac tacccattta tatattatac 540ttatccactt atttaatgtc tttataaggt ttgatccatg atatttctaa tattttagtt 600gatatgtata tgaaagggta ctatttgaac tctcttactc tgtataaagg ttggatcatc 660cttaaagtgg gtctatttaa ttttattgct tcttacagat aaaaaaaaaa ttatgagttg 720gtttgataaa atattgaagg atttaaaata ataataaata acatataata tatgtatata 780aatttattat aatataacat ttatctataa aaaagtaaat attgtcataa atctatacaa 840tcgtttagcc ttgctggacg aatctcaatt atttaaacga gagtaaacat atttgacttt 900ttggttattt aacaaattat tatttaacac tatatgaaat tttttttttt atcagcaaag 960aataaaatta aattaaggag gacaatggtg tcccaatcct tatacaacca acttccacaa 1020gaaagtcaag tcagagacaa caaaaaaaca agcaaaggaa attttttaat ttgagttgtc 1080ttgtttgctg cataatttat gcagtaaaac actacacata acccttttag cagtaaagca 1140atggttgacc gtgtgcttag cttcttttat tttatttttt tatcagcaaa gaataaataa 1200aataaaatga gacacttcag ggatgtttca acggatccaa gcttggcgcg ccgttctata 1260gtgtcaccta aatcgtatgt gtatgataca taaggttatg tattaattgt agccgcgttc 1320taacgacaat atgtccatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 1380aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 1440ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 1500accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 1560taatgtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 1620agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 1680aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 1740ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 1800gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 1860aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 1920aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 1980gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga 2040aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 2100aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 2160cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 2220cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 2280tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 2340tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 2400ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 2460atgcaggttg atcagattcg acatcgatct agtaacatag atgacaccgc gcgcgataat 2520ttatcctagt ttgcgcgcta tattttgttt tctatcgcgt attaaatgta taattgcggg 2580actctaatca taaaaaccca tctcataaat aacgtcatgc attacatgtt aattattaca 2640tgcttaacgt aattcaacag aaattatatg ataatcatcg caagaccggc aacaggattc 2700aatcttaaga aactttattg ccaaatgttt gaacgatctg cttcgacgca ctccttcttt 2760aggtacctca ctattccttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg 2820cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac 2880gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg 2940catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca 3000tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg 3060tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt 3120gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg 3180tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct 3240cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca 3300tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg 3360tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat 3420cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg 3480tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc 3540tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt 3600gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat 3660atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca 3720tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga 3780gttcaggctt tttcatggtt taataagaag agaaaagagt tcttttgtta tggctgaagt 3840aatagagaaa tgagctcgag cgtgtcctct ccaaatgaaa tgaacttcct tatatagagg 3900aagggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg gagatgtcac 3960atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg 4020tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaatg atagcctttc 4080ctttatcgca atgatggcat ttgtaggagc caccttcctt ttctactgtc ctttcgatga 4140agtgacagat agctgggcaa tggaatccga ggaggtttcc cgaaattatc ctttgttgaa 4200aagtctcaat agccctttgg tcttctgaga ctgtatcttt gacatttttg gagtagacca 4260gagtgtcgtg ctccaccatg ttgacgaaga ttttcttctt gtcattgagt cgtaaaagac 4320tctgtatgaa ctgttcgcca gtcttcacgg cgagttctgt tagatcctcg atttgaatct 4380tagactccat gcatggcctt agattcagta ggaactacct ttttagagac tccaatctct 4440attacttgcc ttggtttatg aagcaagcct tgaatcgtcc atactggaat agtacttctg 4500atcttgagaa atatgtcttt ctctgtgttc ttgatgcaat tagtcctgaa tcttttgact 4560gcatctttaa ccttcttggg aaggtatttg atctcctgga gattgttact cgggtagatc 4620gtcttgatga gacctgctgc gtaggcctct ctaaccatct gtgggtcagc attctttctg 4680aaattgaaga ggctaacctt ctcattatca gtggtgaaca tagtgtcgtc accttcacct 4740tcgaacttcc ttcctagatc gtaaagatag aggaaatcgt ccattgtaat ctccggggca 4800aaggagatct cttttggggc tggatcactg ctgggccttt tggttcctag cgtgagccag 4860tgggcttttt gctttggtgg gcttgttagg gccttagcaa agctcttggg cttgagttga 4920gcttctcctt tggggatgaa gttcaacctg tctgtttgct gacttgttgt gtacgcgtca 4980gctgctgctc ttgcctctgt aatagtggca aatttcttgt gtgcaactcc gggaacgccg 5040tttgttgccg cctttgtaca accccagtca tcgtatatac cggcatgtgg accgttatac 5100acaacgtagt agttgatatg agggtgttga atacccgatt ctgctctgag aggagcaact 5160gtgctgttaa gctcagattt ttgtgggatt ggaattggat cgatctcgat cccgcgaaat 5220taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac 5280tttaagaagg agatataccc atggaaaagc ctgaactcac cgcgacgtct gtcgagaagt 5340ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat 5400ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg 5460ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga 5520ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc 5580gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc 5640cggtcgcgga ggctatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg 5700gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga 5760ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg 5820tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc 5880tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg 5940tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct 6000tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc 6060cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac 6120tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg 6180acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg 6240cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca 6300gcactcgtcc gagggcaaag gaatagtgag gtacagcttg gatcgatccg gctgctaaca 6360aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc 6420ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat 6480gatcgggcgc gccgtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg 6540atccagagtt tttataagtt attttataca tgaattaatt ttaacttgtg aaaaaaatta 6600ttttcttctt ataagtattt atgacaaagc ttatataaac atagtcttaa tttcactcag 6660aaaaacagag gaggaaaact tgttgtatga agcccggcta tttcatccat tatccatatt 6720tggatcgaaa agagaaggaa agtgtcattt tatatgtgta taaaaagtat ttcatccata 6780agtaatgata agataattgt gtatgtaaca ttattaatgt atttaaatta aaatcataaa 6840ttattttaaa caattcttat tcgttagtga cacgataacg gataagctaa taatatatct 6900atggttttct gtgaacgtgg cagcatattg atgggaatag ctctgcatgt tgaacaagtg 6960gcacggtacc tagcgtgcct tgctcttctt ttgtctaggc ttggtttggt tcgcatcttc 7020cttctcatat aaatcctcca ccacgtcgag ttttctgttc aaattaaatc gttcaacact 7080ggaactcttt gatataatat agaaagagac agagagagag agacagacaa gaagaacaac 7140catgctagag cggccggagc tggtcatctc gctcatcgtc gagtcggcgg ccggagctgg 7200tcatctcgct catcgtcgag tcggcggccg cgtcatcaac tattccaagc tacgtatttg 7260ggagtttgtg gagtacagca agatgatata cctagacggt gatatccaag tttttgacaa 7320cattgaccac ttgtttgact tgcctgataa ctacttctat gcggtgatgg actgtttctg 7380tgagccaact tggggccaca ctaaacaata tcagatcggt tactgccagc agtgccccca 7440taaggttcag tggcccactc actttgggcc caaacctcct ctctatttca atgctggcat 7500gtttgtgtat gagcccaatt tggctactta ccgtgacctc cttcaaacag tccaagtcac 7560ccagcccact tcctttgctg aacaggattt tttgaacatg tacttcaagg acaaatatag 7620gccaattcct aatgtctaca atcttgtgct ggccatgctg tggcgtcacc ctgagaacgt 7680tgagcttgac aaagttaaag tggttcacta ctgtgctgct gggtctaagc cttggaggta 7740cactgggaag tgactcgagg tcatcaatta ctccaagcta cgtatttggg agttcgtgga 7800gtacaagaag acgatatacc tagacggtga catccaagta tttggaaaca tagaccactt 7860gtttgatctg cctgataatt atttctatgc ggtgatggat tgtttctgcg agaagacttg 7920gagccacacc cctcagttcc agattgggta ctgccaacag tgccctgata aggttcaatg 7980gccctctcac tttggttcca aacctcctct atatttcaat gctggcatgt ttgtttatga 8040gcctaatctc gacacctacc gtgatcttct ccaaactgtc caactcacca agcccacttc 8100ttttgctgag caggactttc tcaacatgta cttcaaggac aagtacaagc caataccgaa 8160catgtacaac cttgtgctgg ccatgttgtg gcgtcaccct gaaaatgttg aacttgataa 8220agttcaagtg gttcattact gtgctgctgg gtctaagcct tggaggttca ctgggaagta 8280actgcaggtc atcaactact ccaagctccg tatatgggag tttgtggagt acagcaagat 8340gatatacttg gacggagaca ttgaggtata tgagaacata gaccacctat ttgacctacc 8400tgatggtaac ttttacgctg tgatggattg tttctgcgag aagacatgga gtcacacccc 8460tcagtacaag gtgggttact gccagcaatg cccggagaag gtgcggtggc ccaccgaatt 8520gggtcagccc ccttctcttt acttcaacgc tggcatgttc gtgttcgaac ccaacatcgc 8580cacctatcat gacctattga aaacggtgca agtcaccact cccacctcgt tcgctgaaca 8640agatttcttg aacatgtact tcaaggacat ttacaagcca atccctttaa attacaatct 8700tgtcctcgcc atgctgtggc gccacccgga aaacgttaaa ttagaccaag tcaaggttgt 8760tcactattgc gcagcggggt ccaagccatg gagatatacg gggaagtagc 8810304479DNAArtificialPlasmid pKR57 30ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg 60tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 120gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 180ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 240ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 300gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 360tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 420aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 480aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 540ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 600tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 660agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 720gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 780tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 840acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 900tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 960caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 1020aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 1080aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 1140ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 1200agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 1260atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 1320cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 1380aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 1440cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 1500aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 1560ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 1620gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 1680ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 1740tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 1800tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 1860ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 1920tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 1980agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 2040acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 2100ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 2160gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg 2220acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 2280gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 2340gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 2400tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa 2460ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg 2520cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa 2580gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt 2640tgtaaaacga cggccagtga attcgagctc ggtacccggg gatcctctag acgtacgttg 2700aaacatccct gaagtgtctc attttatttt atttattctt tgctgataaa aaaataaaat 2760aaaagaagct aagcacacgg tcaaccattg ctctactgct aaaagggtta tgtgtagtgt 2820tttactgcat aaattatgca gcaaacaaga caactcaaat taaaaaattt cctttgcttg 2880tttttttgtt gtctctgact tgactttctt gtggaagttg gttgtataag gattgggaca 2940ccattgtcct tcttaattta attttattct ttgctgataa aaaaaaaaat ttcatatagt 3000gttaaataat aatttgttaa ataaccaaaa agtcaaatat gtttactctc gtttaaataa 3060ttgagattcg tccagcaagg ctaaacgatt gtatagattt atgacaatat ttactttttt 3120atagataaat gttatattat aataaattta tatacatata ttatatgtta tttattatta 3180ttttaaatcc ttcaatattt tatcaaacca actcataatt ttttttttat ctgtaagaag 3240caataaaatt aaatagaccc actttaagga tgatccaacc tttatacaga gtaagagagt 3300tcaaatagta ccctttcata tacatatcaa ctaaaatatt agaaatatca tggatcaaac 3360cttataaaga cattaaataa gtggataagt ataatatata aatgggtagt atataatata 3420taaatggata caaacttctc tctttataat tgttatgtct ccttaacatc ctaatataat 3480acataagtgg gtaatatata atatataaat ggagacaaac ttcttccatt ataattgtta 3540tgtcttctta acacttatgt ctcgttcaca atgctaaggt tagaattgtt tagaaagtct 3600tatagtacac atttgttttt gtactatttg aagcattcca taagccgtca cgattcagat 3660gatttataat aataagagga aatttatcat agaacaataa ggtgcataga tagagtgtta 3720atatatcata acatcctttg tttattcata gaagaagtga gatggagctc agttattata 3780ctgttacatg gtcggataca atattccatg ctctccatga gctcttacac ctacatgcat 3840tttagttcat acttgcggcc gcagtatatc ttaaattctt taatacggtg tactaggata 3900ttgaactggt tcttgatgat gaaaacctgg gccgagattg cagctattta tagtcatagg 3960tcttgttaac atgcatggac atttggccac ggggtggcat gcagtttgac gggtgttgaa 4020ataaacaaaa atgaggtggc ggaagagaat acgagtttga ggttgggtta gaaacaacaa 4080atgtgagggc tcatgatggg ttgagttggt gaatgttttg ggctgctcga ttgacacctt 4140tgtgagtacg tgttgttgtg catggctttt ggggtccagt ttttttttct tgacgcggcg 4200atcctgatca gctagtggat aagtgatgtc cactgtgtgt gattgcgttt ttgtttgaat 4260tttatgaact tagacattgc tatgcaaagg atactctcat tgtgttttgt cttcttttgt 4320tccttggctt tttcttatga tccaagagac tagtcagtgt tgtggcattc gagactacca 4380agattaatta tgatggggga aggataagta actgattagt acggactgtt accaaattaa 4440ttaataagcg gcaaatgaag ggcatggatc ggccggcct 4479315010DNAArtificialPlasmid pKR63 31ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 60tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 120gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 180tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 240ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 300cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 360gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 420tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 480agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 540tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 600cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 660ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 720ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 780ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 840ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 900cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 960gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 1020atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 1080ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 1140gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 1200tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 1260ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 1320taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 1380gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 1440gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 1500ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 1560aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 1620gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 1680cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 1740actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 1800caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 1860gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 1920ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 1980caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 2040tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga

2100gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 2160cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 2220ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 2280gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 2340aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 2400catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg 2460taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag 2520ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 2580ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 2640gtgaattcga gctcggtacc cggggatcct ctagagtcga cctgcaggtc ctcgaagaga 2700agggttaata acacattttt taacattttt aacacaaatt ttagttattt aaaaatttat 2760taaaaaattt aaaataagaa gaggaactct ttaaataaat ctaacttaca aaatttatga 2820tttttaataa gttttcacca ataaaaaatg tcataaaaat atgttaaaaa gtatattatc 2880aatattctct ttatgataaa taaaaagaaa aaaaaaataa aagttaagtg aaaatgagat 2940tgaagtgact ttaggtgtgt ataaatatat caaccccgcc aacaatttat ttaatccaaa 3000tatattgaag tatattattc catagccttt atttatttat atatttatta tataaaagct 3060ttatttgttc taggttgttc atgaaatatt tttttggttt tatctccgtt gtaagaaaat 3120catgtgcttt gtgtcgccac tcactattgc agctttttca tgcattggtc agattgacgg 3180ttgattgtat ttttgttttt tatggttttg tgttatgact taagtcttca tctctttatc 3240tcttcatcag gtttgatggt tacctaatat ggtccatggg tacatgcatg gttaaattag 3300gtggccaact ttgttgtgaa cgatagaatt ttttttatat taagtaaact atttttatat 3360tatgaaataa taataaaaaa aatattttat cattattaac aaaatcatat tagttaattt 3420gttaactcta taataaaaga aatactgtaa cattcacatt acatggtaac atctttccac 3480cctttcattt gttttttgtt tgatgacttt ttttcttgtt taaatttatt tcccttcttt 3540taaatttgga atacattatc atcatatata aactaaaata ctaaaaacag gattacacaa 3600atgataaata ataacacaaa tatttataaa tctagctgca atatatttaa actagctata 3660tcgatattgt aaaataaaac tagctgcatt gatactgata aaaaaatatc atgtgctttc 3720tggactgatg atgcagtata cttttgacat tgcctttatt ttatttttca gaaaagcttt 3780cttagttctg ggttcttcat tatttgtttc ccatctccat tgtgaattga atcatttgct 3840tcgtgtcaca aatacaattt agntaggtac atgcattggt cagattcacg gtttattatg 3900tcatgactta agttcatggt agtacattac ctgccacgca tgcattatat tggttagatt 3960tgataggcaa atttggttgt caacaatata aatataaata atgtttttat attacgaaat 4020aacagtgatc aaaacaaaca gttttatctt tattaacaag attttgtttt tgtttgatga 4080cgttttttaa tgtttacgct ttcccccttc ttttgaattt agaacacttt atcatcataa 4140aatcaaatac taaaaaaatt acatatttca taaataataa cacaaatatt tttaaaaaat 4200ctgaaataat aatgaacaat attacatatt atcacgaaaa ttcattaata aaaatattat 4260ataaataaaa tgtaatagta gttatatgta ggaaaaaagt actgcacgca taatatatac 4320aaaaagatta aaatgaacta ttataaataa taacactaaa ttaatggtga atcatatcaa 4380aataatgaaa aagtaaataa aatttgtaat taacttctat atgtattaca cacacaaata 4440ataaataata gtaaaaaaaa ttatgataaa tatttaccat ctcataagat atttaaaata 4500atgataaaaa tatagattat tttttatgca actagctagc caaaaagaga acacgggtat 4560atataaaaag agtaccttta aattctactg tacttccttt attcctgacg tttttatatc 4620aagtggacat acgtgaagat tttaattatc agtctaaata tttcattagc acttaatact 4680tttctgtttt attcctatcc tataagtagt cccgattctc ccaacattgc ttattcacac 4740aactaactaa gaaagtcttc catagccccc caagcggccg cgacacaagt gtgagagtac 4800taaataaatg ctttggttgt acgaaatcat tacactaaat aaaataatca aagcttatat 4860atgccttccg ctaaggccga atgcaaagaa attggttctt tctcgttatc ttttgccact 4920tttactagta cgtattaatt actacttaat catctttgtt tacggctcat tatatccggc 4980cggcctaaag ggcggatccc ccgggctgca 5010325414DNAArtificialPlasmid pDS1 32aattcgagct cggtacccgg ggatcctcta gagtcgacct gcaggtcctc gaagagaagg 60gttaataaca cattttttaa catttttaac acaaatttta gttatttaaa aatttattaa 120aaaatttaaa ataagaagag gaactcttta aataaatcta acttacaaaa tttatgattt 180ttaataagtt ttcaccaata aaaaatgtca taaaaatatg ttaaaaagta tattatcaat 240attctcttta tgataaataa aaagaaaaaa aaaataaaag ttaagtgaaa atgagattga 300agtgacttta ggtgtgtata aatatatcaa ccccgccaac aatttattta atccaaatat 360attgaagtat attattccat agcctttatt tatttatata tttattatat aaaagcttta 420tttgttctag gttgttcatg aaatattttt ttggttttat ctccgttgta agaaaatcat 480gtgctttgtg tcgccactca ctattgcagc tttttcatgc attggtcaga ttgacggttg 540attgtatttt tgttttttat ggttttgtgt tatgacttaa gtcttcatct ctttatctct 600tcatcaggtt tgatggttac ctaatatggt ccatgggtac atgcatggtt aaattaggtg 660gccaactttg ttgtgaacga tagaattttt tttatattaa gtaaactatt tttatattat 720gaaataataa taaaaaaaat attttatcat tattaacaaa atcatattag ttaatttgtt 780aactctataa taaaagaaat actgtaacat tcacattaca tggtaacatc tttccaccct 840ttcatttgtt ttttgtttga tgactttttt tcttgtttaa atttatttcc cttcttttaa 900atttggaata cattatcatc atatataaac taaaatacta aaaacaggat tacacaaatg 960ataaataata acacaaatat ttataaatct agctgcaata tatttaaact agctatatcg 1020atattgtaaa ataaaactag ctgcattgat actgataaaa aaatatcatg tgctttctgg 1080actgatgatg cagtatactt ttgacattgc ctttatttta tttttcagaa aagctttctt 1140agttctgggt tcttcattat ttgtttccca tctccattgt gaattgaatc atttgcttcg 1200tgtcacaaat acaatttagn taggtacatg cattggtcag attcacggtt tattatgtca 1260tgacttaagt tcatggtagt acattacctg ccacgcatgc attatattgg ttagatttga 1320taggcaaatt tggttgtcaa caatataaat ataaataatg tttttatatt acgaaataac 1380agtgatcaaa acaaacagtt ttatctttat taacaagatt ttgtttttgt ttgatgacgt 1440tttttaatgt ttacgctttc ccccttcttt tgaatttaga acactttatc atcataaaat 1500caaatactaa aaaaattaca tatttcataa ataataacac aaatattttt aaaaaatctg 1560aaataataat gaacaatatt acatattatc acgaaaattc attaataaaa atattatata 1620aataaaatgt aatagtagtt atatgtagga aaaaagtact gcacgcataa tatatacaaa 1680aagattaaaa tgaactatta taaataataa cactaaatta atggtgaatc atatcaaaat 1740aatgaaaaag taaataaaat ttgtaattaa cttctatatg tattacacac acaaataata 1800aataatagta aaaaaaatta tgataaatat ttaccatctc ataagatatt taaaataatg 1860ataaaaatat agattatttt ttatgcaact agctagccaa aaagagaaca cgggtatata 1920taaaaagagt acctttaaat tctactgtac ttcctttatt cctgacgttt ttatatcaag 1980tggacatacg tgaagatttt aattatcagt ctaaatattt cattagcact taatactttt 2040ctgttttatt cctatcctat aagtagtccc gattctccca acattgctta ttcacacaac 2100taactaagaa agtcttccat agccccccaa gcggccgcag tatatcttaa attctttaat 2160acggtgtact aggatattga actggttctt gatgatgaaa acctgggccg agattgcagc 2220tatttatagt cataggtctt gttaacatgc atggacattt ggccacgggg tggcatgcag 2280tttgacgggt gttgaaataa acaaaaatga ggtggcggaa gagaatacga gtttgaggtt 2340gggttagaaa caacaaatgt gagggctcat gatgggttga gttggtgaat gttttgggct 2400gctcgattga cacctttgtg agtacgtgtt gttgtgcatg gcttttgggg tccagttttt 2460ttttcttgac gcggcgatcc tgatcagcta gtggataagt gatgtccact gtgtgtgatt 2520gcgtttttgt ttgaatttta tgaacttaga cattgctatg caaaggatac tctcattgtg 2580ttttgtcttc ttttgttcct tggctttttc ttatgatcca agagactagt cagtgttgtg 2640gcattcgaga ctaccaagat taattatgat gggggaagga taagtaactg attagtacgg 2700actgttacca aattaattaa taagcggcaa atgaagggca tggatcggcc ggcctctaga 2760gtcgacctgc aggcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 2820ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 2880gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 2940gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 3000tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3060gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 3120ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3180ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 3240acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 3300tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3360ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 3420ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3480ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 3540actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 3600gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 3660tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 3720caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 3780atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 3840acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 3900ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 3960ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 4020tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 4080tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 4140gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 4200tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4260tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 4320ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4380tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 4440ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 4500gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 4560ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 4620cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4680ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 4740ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 4800gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 4860ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 4920gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 4980aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 5040tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 5100cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 5160taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 5220gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa 5280ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 5340atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 5400aacgacggcc agtg 5414337085DNAArtificialPlasmid pKR72 33gtacggatcc gtcgacggcg cgcccgatca tccggatata gttcctcctt tcagcaaaaa 60acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc 120agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcgatcc aagctgtacc 180tcactattcc tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac 240ttctacacag ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac 300agtcccggct ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc 360gaaattgccg tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc 420ccggagccgc ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg 480ctccatacaa gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc 540cccgaacatc gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac 600attgttggag ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca 660aagcatcagc tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt 720ttgccagtga tacacatggg gatcagcaat cgcgcatatg aaatcacgcc atgtagtgta 780ttgaccgatt ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc 840agcgatcgca tccatagcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg 900caggtcttgc aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct 960gaattcccca atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgata 1020aacataacga tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg 1080ccctcctaca tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc 1140ggagacgctg tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg 1200cttttccatg ggtatatctc cttcttaaag ttaaacaaaa ttatttctag agggaaaccg 1260ttgtggtctc cctatagtga gtcgtattaa tttcgcggga tcgagatcga tccaattcca 1320atcccacaaa aatctgagct taacagcaca gttgctcctc tcagagcaga atcgggtatt 1380caacaccctc atatcaacta ctacgttgtg tataacggtc cacatgccgg tatatacgat 1440gactggggtt gtacaaaggc ggcaacaaac ggcgttcccg gagttgcaca caagaaattt 1500gccactatta cagaggcaag agcagcagct gacgcgtaca caacaagtca gcaaacagac 1560aggttgaact tcatccccaa aggagaagct caactcaagc ccaagagctt tgctaaggcc 1620ctaacaagcc caccaaagca aaaagcccac tggctcacgc taggaaccaa aaggcccagc 1680agtgatccag ccccaaaaga gatctccttt gccccggaga ttacaatgga cgatttcctc 1740tatctttacg atctaggaag gaagttcgaa ggtgaaggtg acgacactat gttcaccact 1800gataatgaga aggttagcct cttcaatttc agaaagaatg ctgacccaca gatggttaga 1860gaggcctacg cagcaggtct catcaagacg atctacccga gtaacaatct ccaggagatc 1920aaataccttc ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ttgcatcaag 1980aacacagaga aagacatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa 2040ggcttgcttc ataaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttcct 2100actgaatcta aggccatgca tggagtctaa gattcaaatc gaggatctaa cagaactcgc 2160cgtgaagact ggcgaacagt tcatacagag tcttttacga ctcaatgaca agaagaaaat 2220cttcgtcaac atggtggagc acgacactct ggtctactcc aaaaatgtca aagatacagt 2280ctcagaagac caaagggcta ttgagacttt tcaacaaagg ataatttcgg gaaacctcct 2340cggattccat tgcccagcta tctgtcactt catcgaaagg acagtagaaa aggaaggtgg 2400ctcctacaaa tgccatcatt gcgataaagg aaaggctatc attcaagatg cctctgccga 2460cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc 2520aaccacgtct tcaaagcaag tggattgatg tgacatctcc actgacgtaa gggatgacgc 2580acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat ttcatttgga 2640gaggacacgc tcgagctcat ttctctatta cttcagccat aacaaaagaa ctcttttctc 2700ttcttattaa accatgaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat 2760cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc 2820tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg 2880tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga 2940agtgcttgac attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca 3000gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc 3060ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt 3120cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga 3180tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca 3240ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca 3300cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga 3360ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag 3420gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct 3480tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca 3540gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat 3600cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt 3660ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg 3720tccgagggca aaggaatagt gaggtaccta aagaaggagt gcgtcgaagc agatcgttca 3780aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 3840atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 3900tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 3960aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 4020gatcgatgtc gaatcgatca acctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4080tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4140gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4200ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4260ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4320acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4380tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4440ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 4500ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4560ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4620actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 4680gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 4740tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4800caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4860atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4920acgttaaggg attttggtca tgacattaac ctataaaaat aggcgtatca cgaggccctt 4980tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac 5040ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc 5100gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag 5160agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg 5220tatcatacac atacgattta ggtgacacta tagaacggcg cgccaagctt gttgaaacat 5280ccctgaagtg tctcatttta ttttatttat tctttgctga taaaaaaata aaataaaaga 5340agctaagcac acggtcaacc attgctctac tgctaaaagg gttatgtgta gtgttttact 5400gcataaatta tgcagcaaac aagacaactc aaattaaaaa atttcctttg cttgtttttt 5460tgttgtctct gacttgactt tcttgtggaa gttggttgta taaggattgg gacaccattg 5520tccttcttaa tttaatttta ttctttgctg ataaaaaaaa aaatttcata tagtgttaaa 5580taataatttg ttaaataacc aaaaagtcaa atatgtttac tctcgtttaa ataattgaga 5640ttcgtccagc aaggctaaac gattgtatag atttatgaca atatttactt ttttatagat 5700aaatgttata ttataataaa tttatataca tatattatat gttatttatt attattttaa 5760atccttcaat attttatcaa accaactcat aatttttttt ttatctgtaa gaagcaataa 5820aattaaatag acccacttta aggatgatcc aacctttata cagagtaaga gagttcaaat 5880agtacccttt catatacata tcaactaaaa tattagaaat atcatggatc aaaccttata 5940aagacattaa ataagtggat aagtataata tataaatggg tagtatataa tatataaatg 6000gatacaaact tctctcttta taattgttat gtctccttaa catcctaata taatacataa 6060gtgggtaata tataatatat aaatggagac aaacttcttc cattataatt gttatgtctt 6120cttaacactt atgtctcgtt cacaatgcta aggttagaat tgtttagaaa gtcttatagt 6180acacatttgt ttttgtacta tttgaagcat tccataagcc gtcacgattc agatgattta 6240taataataag aggaaattta tcatagaaca ataaggtgca tagatagagt gttaatatat 6300cataacatcc tttgtttatt catagaagaa gtgagatgga gctcagttat tatactgtta 6360catggtcgga tacaatattc catgctctcc atgagctctt acacctacat gcattttagt 6420tcatacttgc ggccgcagta tatcttaaat tctttaatac ggtgtactag gatattgaac 6480tggttcttga tgatgaaaac ctgggccgag attgcagcta tttatagtca taggtcttgt 6540taacatgcat ggacatttgg ccacggggtg gcatgcagtt tgacgggtgt tgaaataaac

6600aaaaatgagg tggcggaaga gaatacgagt ttgaggttgg gttagaaaca acaaatgtga 6660gggctcatga tgggttgagt tggtgaatgt tttgggctgc tcgattgaca cctttgtgag 6720tacgtgttgt tgtgcatggc ttttggggtc cagttttttt ttcttgacgc ggcgatcctg 6780atcagctagt ggataagtga tgtccactgt gtgtgattgc gtttttgttt gaattttatg 6840aacttagaca ttgctatgca aaggatactc tcattgtgtt ttgtcttctt ttgttccttg 6900gctttttctt atgatccaag agactagtca gtgttgtggc attcgagact accaagatta 6960attatgatgg gggaaggata agtaactgat tagtacggac tgttaccaaa ttaattaata 7020agcggcaaat gaagggcatg gatcaaaagc ttggatctcc tgcaggatct ggccggccgg 7080atctc 7085345303DNAArtificialPlasmid pDS2 34agcttggatc tcctgcagga tctggccggc cggatctcgt acggatccgt cgacggcgcg 60cccgatcatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag 120gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt 180cgggctttgt tagcagccgg atcgatccaa gctgtacctc actattcctt tgccctcgga 240cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag 300acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg 360attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc 420tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg cgatcctgca 480agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc 540ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag 600tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg 660tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 720tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga 780tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg 840aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catagcctcc 900gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc 960tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat gtcaagcact 1020tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa 1080ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa 1140gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg 1200atcagaaact tctcgacaga cgtcgcggtg agttcaggct tttccatggg tatatctcct 1260tcttaaagtt aaacaaaatt atttctagag ggaaaccgtt gtggtctccc tatagtgagt 1320cgtattaatt tcgcgggatc gagatcgatc caattccaat cccacaaaaa tctgagctta 1380acagcacagt tgctcctctc agagcagaat cgggtattca acaccctcat atcaactact 1440acgttgtgta taacggtcca catgccggta tatacgatga ctggggttgt acaaaggcgg 1500caacaaacgg cgttcccgga gttgcacaca agaaatttgc cactattaca gaggcaagag 1560cagcagctga cgcgtacaca acaagtcagc aaacagacag gttgaacttc atccccaaag 1620gagaagctca actcaagccc aagagctttg ctaaggccct aacaagccca ccaaagcaaa 1680aagcccactg gctcacgcta ggaaccaaaa ggcccagcag tgatccagcc ccaaaagaga 1740tctcctttgc cccggagatt acaatggacg atttcctcta tctttacgat ctaggaagga 1800agttcgaagg tgaaggtgac gacactatgt tcaccactga taatgagaag gttagcctct 1860tcaatttcag aaagaatgct gacccacaga tggttagaga ggcctacgca gcaggtctca 1920tcaagacgat ctacccgagt aacaatctcc aggagatcaa ataccttccc aagaaggtta 1980aagatgcagt caaaagattc aggactaatt gcatcaagaa cacagagaaa gacatatttc 2040tcaagatcag aagtactatt ccagtatgga cgattcaagg cttgcttcat aaaccaaggc 2100aagtaataga gattggagtc tctaaaaagg tagttcctac tgaatctaag gccatgcatg 2160gagtctaaga ttcaaatcga ggatctaaca gaactcgccg tgaagactgg cgaacagttc 2220atacagagtc ttttacgact caatgacaag aagaaaatct tcgtcaacat ggtggagcac 2280gacactctgg tctactccaa aaatgtcaaa gatacagtct cagaagacca aagggctatt 2340gagacttttc aacaaaggat aatttcggga aacctcctcg gattccattg cccagctatc 2400tgtcacttca tcgaaaggac agtagaaaag gaaggtggct cctacaaatg ccatcattgc 2460gataaaggaa aggctatcat tcaagatgcc tctgccgaca gtggtcccaa agatggaccc 2520ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 2580gattgatgtg acatctccac tgacgtaagg gatgacgcac aatcccacta tccttcgcaa 2640gacccttcct ctatataagg aagttcattt catttggaga ggacacgctc gagctcattt 2700ctctattact tcagccataa caaaagaact cttttctctt cttattaaac catgaaaaag 2760cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc 2820gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg 2880cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt 2940tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc 3000agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg 3060cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct 3120gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa 3180tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa 3240actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt 3300tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat 3360gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg 3420gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag 3480cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg 3540gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc 3600gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact 3660gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa 3720gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaatagtga 3780ggtacctaaa gaaggagtgc gtcgaagcag atcgttcaaa catttggcaa taaagtttct 3840taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg 3900ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga 3960ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact 4020aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgatgtcga atcgatcaac 4080ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 4140gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 4200cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 4260tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 4320cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 4380aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 4440cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 4500gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 4560ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 4620cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 4680aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 4740tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 4800ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 4860tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 4920ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 4980acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat 5040gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg 5100gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc 5160tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg 5220tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg 5280tgacactata gaacggcgcg cca 5303358031DNAArtificialPlasmid pDS3 (orientation 2) 35tcgactctag aggccggccg atccatgccc ttcatttgcc gcttattaat taatttggta 60acagtccgta ctaatcagtt acttatcctt cccccatcat aattaatctt ggtagtctcg 120aatgccacaa cactgactag tctcttggat cataagaaaa agccaaggaa caaaagaaga 180caaaacacaa tgagagtatc ctttgcatag caatgtctaa gttcataaaa ttcaaacaaa 240aacgcaatca cacacagtgg acatcactta tccactagct gatcaggatc gccgcgtcaa 300gaaaaaaaaa ctggacccca aaagccatgc acaacaacac gtactcacaa aggtgtcaat 360cgagcagccc aaaacattca ccaactcaac ccatcatgag ccctcacatt tgttgtttct 420aacccaacct caaactcgta ttctcttccg ccacctcatt tttgtttatt tcaacacccg 480tcaaactgca tgccaccccg tggccaaatg tccatgcatg ttaacaagac ctatgactat 540aaatagctgc aatctcggcc caggttttca tcatcaagaa ccagttcaat atcctagtac 600accgtattaa agaatttaag atatactgcg gccgcttggg gggctatgga agactttctt 660agttagttgt gtgaataagc aatgttggga gaatcgggac tacttatagg ataggaataa 720aacagaaaag tattaagtgc taatgaaata tttagactga taattaaaat cttcacgtat 780gtccacttga tataaaaacg tcaggaataa aggaagtaca gtagaattta aaggtactct 840ttttatatat acccgtgttc tctttttggc tagctagttg cataaaaaat aatctatatt 900tttatcatta ttttaaatat cttatgagat ggtaaatatt tatcataatt ttttttacta 960ttatttatta tttgtgtgtg taatacatat agaagttaat tacaaatttt atttactttt 1020tcattatttt gatatgattc accattaatt tagtgttatt atttataata gttcatttta 1080atctttttgt atatattatg cgtgcagtac ttttttccta catataacta ctattacatt 1140ttatttatat aatattttta ttaatgaatt ttcgtgataa tatgtaatat tgttcattat 1200tatttcagat tttttaaaaa tatttgtgtt attatttatg aaatatgtaa tttttttagt 1260atttgatttt atgatgataa agtgttctaa attcaaaaga agggggaaag cgtaaacatt 1320aaaaaacgtc atcaaacaaa aacaaaatct tgttaataaa gataaaactg tttgttttga 1380tcactgttat ttcgtaatat aaaaacatta tttatattta tattgttgac aaccaaattt 1440gcctatcaaa tctaaccaat ataatgcatg cgtggcaggt aatgtactac catgaactta 1500agtcatgaca taataaaccg tgaatctgac caatgcatgt acctanctaa attgtatttg 1560tgacacgaag caaatgattc aattcacaat ggagatggga aacaaataat gaagaaccca 1620gaactaagaa agcttttctg aaaaataaaa taaaggcaat gtcaaaagta tactgcatca 1680tcagtccaga aagcacatga tattttttta tcagtatcaa tgcagctagt tttattttac 1740aatatcgata tagctagttt aaatatattg cagctagatt tataaatatt tgtgttatta 1800tttatcattt gtgtaatcct gtttttagta ttttagttta tatatgatga taatgtattc 1860caaatttaaa agaagggaaa taaatttaaa caagaaaaaa agtcatcaaa caaaaaacaa 1920atgaaagggt ggaaagatgt taccatgtaa tgtgaatgtt acagtatttc ttttattata 1980gagttaacaa attaactaat atgattttgt taataatgat aaaatatttt ttttattatt 2040atttcataat ataaaaatag tttacttaat ataaaaaaaa ttctatcgtt cacaacaaag 2100ttggccacct aatttaacca tgcatgtacc catggaccat attaggtaac catcaaacct 2160gatgaagaga taaagagatg aagacttaag tcataacaca aaaccataaa aaacaaaaat 2220acaatcaacc gtcaatctga ccaatgcatg aaaaagctgc aatagtgagt ggcgacacaa 2280agcacatgat tttcttacaa cggagataaa accaaaaaaa tatttcatga acaacctaga 2340acaaataaag cttttatata ataaatatat aaataaataa aggctatgga ataatatact 2400tcaatatatt tggattaaat aaattgttgg cggggttgat atatttatac acacctaaag 2460tcacttcaat ctcattttca cttaactttt attttttttt tctttttatt tatcataaag 2520agaatattga taatatactt tttaacatat ttttatgaca ttttttattg gtgaaaactt 2580attaaaaatc ataaattttg taagttagat ttatttaaag agttcctctt cttattttaa 2640attttttaat aaatttttaa ataactaaaa tttgtgttaa aaatgttaaa aaatgtgtta 2700ttaacccttc tcttcgagga cctgcaggtc gacggcgcgc ccgatcatcc ggatatagtt 2760cctcctttca gcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctagt 2820tattgctcag cggtggcagc agccaactca gcttcctttc gggctttgtt agcagccgga 2880tcgatccaag ctgtacctca ctattccttt gccctcggac gagtgctggg gcgtcggttt 2940ccactatcgg cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgcgggcg 3000atttgtgtac gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc 3060gcccaagctg catcatcgaa attgccgtca accaagctct gatagagttg gtcaagacca 3120atgcggagca tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg 3180aagtagcgcg tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg 3240acctcgtatt gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg 3300ccattgtccg tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg 3360gggcagtcct cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg 3420gtgtcgtcca tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa 3480tcacgccatg tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc 3540tggctaagat cggccgcagc gatcgcatcc atagcctccg cgaccggctg cagaacagcg 3600ggcagttcgg tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa 3660taggtcaggc tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc 3720gatgcaaagt gccgataaac ataacgatct ttgtagaaac catcggcgca gctatttacc 3780cgcaggacat atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc 3840gagagctgca tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac 3900gtcgcggtga gttcaggctt ttccatgggt atatctcctt cttaaagtta aacaaaatta 3960tttctagagg gaaaccgttg tggtctccct atagtgagtc gtattaattt cgcgggatcg 4020agatcgatcc aattccaatc ccacaaaaat ctgagcttaa cagcacagtt gctcctctca 4080gagcagaatc gggtattcaa caccctcata tcaactacta cgttgtgtat aacggtccac 4140atgccggtat atacgatgac tggggttgta caaaggcggc aacaaacggc gttcccggag 4200ttgcacacaa gaaatttgcc actattacag aggcaagagc agcagctgac gcgtacacaa 4260caagtcagca aacagacagg ttgaacttca tccccaaagg agaagctcaa ctcaagccca 4320agagctttgc taaggcccta acaagcccac caaagcaaaa agcccactgg ctcacgctag 4380gaaccaaaag gcccagcagt gatccagccc caaaagagat ctcctttgcc ccggagatta 4440caatggacga tttcctctat ctttacgatc taggaaggaa gttcgaaggt gaaggtgacg 4500acactatgtt caccactgat aatgagaagg ttagcctctt caatttcaga aagaatgctg 4560acccacagat ggttagagag gcctacgcag caggtctcat caagacgatc tacccgagta 4620acaatctcca ggagatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca 4680ggactaattg catcaagaac acagagaaag acatatttct caagatcaga agtactattc 4740cagtatggac gattcaaggc ttgcttcata aaccaaggca agtaatagag attggagtct 4800ctaaaaaggt agttcctact gaatctaagg ccatgcatgg agtctaagat tcaaatcgag 4860gatctaacag aactcgccgt gaagactggc gaacagttca tacagagtct tttacgactc 4920aatgacaaga agaaaatctt cgtcaacatg gtggagcacg acactctggt ctactccaaa 4980aatgtcaaag atacagtctc agaagaccaa agggctattg agacttttca acaaaggata 5040atttcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat cgaaaggaca 5100gtagaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggctatcatt 5160caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 5220gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga catctccact 5280gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataagga 5340agttcatttc atttggagag gacacgctcg agctcatttc tctattactt cagccataac 5400aaaagaactc ttttctcttc ttattaaacc atgaaaaagc ctgaactcac cgcgacgtct 5460gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag 5520ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta 5580aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc 5640gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc 5700atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct 5760gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg 5820agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc 5880atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc 5940agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa 6000gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc 6060ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc 6120aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag 6180cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt 6240cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag 6300ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc 6360cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac 6420cgacgcccca gcactcgtcc gagggcaaag gaatagtgag gtacctaaag aaggagtgcg 6480tcgaagcaga tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg 6540gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca 6600tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg caattataca 6660tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg 6720tgtcatctat gttactagat cgatgtcgaa tcgatcaacc tgcattaatg aatcggccaa 6780cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 6840ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 6900ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 6960gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 7020gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 7080taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 7140accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 7200tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 7260cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 7320agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 7380gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 7440gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 7500tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 7560acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 7620cagtggaacg aaaactcacg ttaagggatt ttggtcatga cattaaccta taaaaatagg 7680cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 7740atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 7800cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 7860gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat 7920taatacataa ccttatgtat catacacata cgatttaggt gacactatag aacggcgcgc 7980caagcttgga tctcctgcag gatctggccg gccggatctc gtacggatcc g 8031369616DNAArtificialPLasmid SH60 36ggccgcgtca tcaactattc caagctacgt atttgggagt ttgtggagta cagcaagatg 60atatacctag acggtgatat ccaagttttt gacaacattg accacttgtt tgacttgcct 120gataactact tctatgcggt gatggactgt ttctgtgagc caacttgggg ccacactaaa 180caatatcaga tcggttactg ccagcagtgc ccccataagg ttcagtggcc cactcacttt 240gggcccaaac ctcctctcta tttcaatgct ggcatgtttg tgtatgagcc caatttggct 300acttaccgtg acctccttca aacagtccaa gtcacccagc ccacttcctt tgctgaacag 360gattttttga acatgtactt caaggacaaa tataggccaa ttcctaatgt ctacaatctt 420gtgctggcca tgctgtggcg tcaccctgag aacgttgagc ttgacaaagt taaagtggtt 480cactactgtg ctgctgggtc taagccttgg aggtacactg ggaagtgact cgaggtcatc 540aattactcca agctacgtat ttgggagttc gtggagtaca agaagacgat atacctagac 600ggtgacatcc aagtatttgg aaacatagac cacttgtttg atctgcctga taattatttc 660tatgcggtga tggattgttt ctgcgagaag acttggagcc acacccctca gttccagatt 720gggtactgcc aacagtgccc tgataaggtt caatggccct ctcactttgg ttccaaacct 780cctctatatt tcaatgctgg catgtttgtt tatgagccta atctcgacac ctaccgtgat 840cttctccaaa ctgtccaact caccaagccc acttcttttg ctgagcagga ctttctcaac 900atgtacttca aggacaagta caagccaata ccgaacatgt acaaccttgt gctggccatg 960ttgtggcgtc accctgaaaa tgttgaactt gataaagttc aagtggttca ttactgtgct 1020gctgggtcta agccttggag

gttcactggg aagtaactgc aggtcatcaa ctactccaag 1080ctccgtatat gggagtttgt ggagtacagc aagatgatat acttggacgg agacattgag 1140gtatatgaga acatagacca cctatttgac ctacctgatg gtaactttta cgctgtgatg 1200gattgtttct gcgagaagac atggagtcac acccctcagt acaaggtggg ttactgccag 1260caatgcccgg agaaggtgcg gtggcccacc gaattgggtc agcccccttc tctttacttc 1320aacgctggca tgttcgtgtt cgaacccaac atcgccacct atcatgacct attgaaaacg 1380gtgcaagtca ccactcccac ctcgttcgct gaacaagatt tcttgaacat gtacttcaag 1440gacatttaca agccaatccc tttaaattac aatcttgtcc tcgccatgct gtggcgccac 1500ccggaaaacg ttaaattaga ccaagtcaag gttgttcact attgcgcagc ggggtccaag 1560ccatggagat atacggggaa gtagcggccg cttggggggc tatggaagac tttcttagtt 1620agttgtgtga ataagcaatg ttgggagaat cgggactact tataggatag gaataaaaca 1680gaaaagtatt aagtgctaat gaaatattta gactgataat taaaatcttc acgtatgtcc 1740acttgatata aaaacgtcag gaataaagga agtacagtag aatttaaagg tactcttttt 1800atatataccc gtgttctctt tttggctagc tagttgcata aaaaataatc tatattttta 1860tcattatttt aaatatctta tgagatggta aatatttatc ataatttttt ttactattat 1920ttattatttg tgtgtgtaat acatatagaa gttaattaca aattttattt actttttcat 1980tattttgata tgattcacca ttaatttagt gttattattt ataatagttc attttaatct 2040ttttgtatat attatgcgtg cagtactttt ttcctacata taactactat tacattttat 2100ttatataata tttttattaa tgaattttcg tgataatatg taatattgtt cattattatt 2160tcagattttt taaaaatatt tgtgttatta tttatgaaat atgtaatttt tttagtattt 2220gattttatga tgataaagtg ttctaaattc aaaagaaggg ggaaagcgta aacattaaaa 2280aacgtcatca aacaaaaaca aaatcttgtt aataaagata aaactgtttg ttttgatcac 2340tgttatttcg taatataaaa acattattta tatttatatt gttgacaacc aaatttgcct 2400atcaaatcta accaatataa tgcatgcgtg gcaggtaatg tactaccatg aacttaagtc 2460atgacataat aaaccgtgaa tctgaccaat gcatgtacct anctaaattg tatttgtgac 2520acgaagcaaa tgattcaatt cacaatggag atgggaaaca aataatgaag aacccagaac 2580taagaaagct tttctgaaaa ataaaataaa ggcaatgtca aaagtatact gcatcatcag 2640tccagaaagc acatgatatt tttttatcag tatcaatgca gctagtttta ttttacaata 2700tcgatatagc tagtttaaat atattgcagc tagatttata aatatttgtg ttattattta 2760tcatttgtgt aatcctgttt ttagtatttt agtttatata tgatgataat gtattccaaa 2820tttaaaagaa gggaaataaa tttaaacaag aaaaaaagtc atcaaacaaa aaacaaatga 2880aagggtggaa agatgttacc atgtaatgtg aatgttacag tatttctttt attatagagt 2940taacaaatta actaatatga ttttgttaat aatgataaaa tatttttttt attattattt 3000cataatataa aaatagttta cttaatataa aaaaaattct atcgttcaca acaaagttgg 3060ccacctaatt taaccatgca tgtacccatg gaccatatta ggtaaccatc aaacctgatg 3120aagagataaa gagatgaaga cttaagtcat aacacaaaac cataaaaaac aaaaatacaa 3180tcaaccgtca atctgaccaa tgcatgaaaa agctgcaata gtgagtggcg acacaaagca 3240catgattttc ttacaacgga gataaaacca aaaaaatatt tcatgaacaa cctagaacaa 3300ataaagcttt tatataataa atatataaat aaataaaggc tatggaataa tatacttcaa 3360tatatttgga ttaaataaat tgttggcggg gttgatatat ttatacacac ctaaagtcac 3420ttcaatctca ttttcactta acttttattt tttttttctt tttatttatc ataaagagaa 3480tattgataat atacttttta acatattttt atgacatttt ttattggtga aaacttatta 3540aaaatcataa attttgtaag ttagatttat ttaaagagtt cctcttctta ttttaaattt 3600tttaataaat ttttaaataa ctaaaatttg tgttaaaaat gttaaaaaat gtgttattaa 3660cccttctctt cgaggacctg caggtcgacg gcgcgcccga tcatccggat atagttcctc 3720ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat gctagttatt 3780gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgttagca gccggatcga 3840tccaagctgt acctcactat tcctttgccc tcggacgagt gctggggcgt cggtttccac 3900tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt 3960gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc 4020aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc 4080ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc cgctcgaagt 4140agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct 4200cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat 4260tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc 4320agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt 4380cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac 4440gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc 4500taagatcggc cgcagcgatc gcatccatag cctccgcgac cggctgcaga acagcgggca 4560gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg 4620tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg 4680caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca 4740ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga 4800gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg 4860cggtgagttc aggcttttcc atgggtatat ctccttctta aagttaaaca aaattatttc 4920tagagggaaa ccgttgtggt ctccctatag tgagtcgtat taatttcgcg ggatcgagat 4980cgatccaatt ccaatcccac aaaaatctga gcttaacagc acagttgctc ctctcagagc 5040agaatcgggt attcaacacc ctcatatcaa ctactacgtt gtgtataacg gtccacatgc 5100cggtatatac gatgactggg gttgtacaaa ggcggcaaca aacggcgttc ccggagttgc 5160acacaagaaa tttgccacta ttacagaggc aagagcagca gctgacgcgt acacaacaag 5220tcagcaaaca gacaggttga acttcatccc caaaggagaa gctcaactca agcccaagag 5280ctttgctaag gccctaacaa gcccaccaaa gcaaaaagcc cactggctca cgctaggaac 5340caaaaggccc agcagtgatc cagccccaaa agagatctcc tttgccccgg agattacaat 5400ggacgatttc ctctatcttt acgatctagg aaggaagttc gaaggtgaag gtgacgacac 5460tatgttcacc actgataatg agaaggttag cctcttcaat ttcagaaaga atgctgaccc 5520acagatggtt agagaggcct acgcagcagg tctcatcaag acgatctacc cgagtaacaa 5580tctccaggag atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac 5640taattgcatc aagaacacag agaaagacat atttctcaag atcagaagta ctattccagt 5700atggacgatt caaggcttgc ttcataaacc aaggcaagta atagagattg gagtctctaa 5760aaaggtagtt cctactgaat ctaaggccat gcatggagtc taagattcaa atcgaggatc 5820taacagaact cgccgtgaag actggcgaac agttcataca gagtctttta cgactcaatg 5880acaagaagaa aatcttcgtc aacatggtgg agcacgacac tctggtctac tccaaaaatg 5940tcaaagatac agtctcagaa gaccaaaggg ctattgagac ttttcaacaa aggataattt 6000cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag 6060aaaaggaagg tggctcctac aaatgccatc attgcgataa aggaaaggct atcattcaag 6120atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 6180aagaagacgt tccaaccacg tcttcaaagc aagtggattg atgtgacatc tccactgacg 6240taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt 6300catttcattt ggagaggaca cgctcgagct catttctcta ttacttcagc cataacaaaa 6360gaactctttt ctcttcttat taaaccatga aaaagcctga actcaccgcg acgtctgtcg 6420agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 6480aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 6540gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 6600tcccgattcc ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct 6660cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 6720tgcagccggt cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg 6780ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 6840gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 6900cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 6960ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 7020cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 7080tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 7140ggcatccgga gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg 7200accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 7260gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 7320gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 7380gccccagcac tcgtccgagg gcaaaggaat agtgaggtac ctaaagaagg agtgcgtcga 7440agcagatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 7500tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 7560atgcatgacg ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 7620atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 7680atctatgtta ctagatcgat gtcgaatcga tcaacctgca ttaatgaatc ggccaacgcg 7740cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 7800gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 7860ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 7920ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 7980atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 8040aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 8100gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 8160ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 8220ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 8280acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 8340gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 8400ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 8460ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 8520gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 8580ggaacgaaaa ctcacgttaa gggattttgg tcatgacatt aacctataaa aataggcgta 8640tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc 8700agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc 8760agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc 8820agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat 8880acataacctt atgtatcata cacatacgat ttaggtgaca ctatagaacg gcgcgccaag 8940cttggatctc ctgcaggatc tggccggccg gatctcgtac ggatccgtcg actctagagg 9000ccggccgatc catgcccttc atttgccgct tattaattaa tttggtaaca gtccgtacta 9060atcagttact tatccttccc ccatcataat taatcttggt agtctcgaat gccacaacac 9120tgactagtct cttggatcat aagaaaaagc caaggaacaa aagaagacaa aacacaatga 9180gagtatcctt tgcatagcaa tgtctaagtt cataaaattc aaacaaaaac gcaatcacac 9240acagtggaca tcacttatcc actagctgat caggatcgcc gcgtcaagaa aaaaaaactg 9300gaccccaaaa gccatgcaca acaacacgta ctcacaaagg tgtcaatcga gcagcccaaa 9360acattcacca actcaaccca tcatgagccc tcacatttgt tgtttctaac ccaacctcaa 9420actcgtattc tcttccgcca cctcattttt gtttatttca acacccgtca aactgcatgc 9480caccccgtgg ccaaatgtcc atgcatgtta acaagaccta tgactataaa tagctgcaat 9540ctcggcccag gttttcatca tcaagaacca gttcaatatc ctagtacacc gtattaaaga 9600atttaagata tactgc 9616371585DNAArtificialNot1 fragment 37ggccgcgtca tcaactattc caagctacgt atttgggagt ttgtggagta cagcaagatg 60atatacctag acggtgatat ccaagttttt gacaacattg accacttgtt tgacttgcct 120gataactact tctatgcggt gatggactgt ttctgtgagc caacttgggg ccacactaaa 180caatatcaga tcggttactg ccagcagtgc ccccataagg ttcagtggcc cactcacttt 240gggcccaaac ctcctctcta tttcaatgct ggcatgtttg tgtatgagcc caatttggct 300acttaccgtg acctccttca aacagtccaa gtcacccagc ccacttcctt tgctgaacag 360gattttttga acatgtactt caaggacaaa tataggccaa ttcctaatgt ctacaatctt 420gtgctggcca tgctgtggcg tcaccctgag aacgttgagc ttgacaaagt taaagtggtt 480cactactgtg ctgctgggtc taagccttgg aggtacactg ggaagtgact cgaggtcatc 540aattactcca agctacgtat ttgggagttc gtggagtaca agaagacgat atacctagac 600ggtgacatcc aagtatttgg aaacatagac cacttgtttg atctgcctga taattatttc 660tatgcggtga tggattgttt ctgcgagaag acttggagcc acacccctca gttccagatt 720gggtactgcc aacagtgccc tgataaggtt caatggccct ctcactttgg ttccaaacct 780cctctatatt tcaatgctgg catgtttgtt tatgagccta atctcgacac ctaccgtgat 840cttctccaaa ctgtccaact caccaagccc acttcttttg ctgagcagga ctttctcaac 900atgtacttca aggacaagta caagccaata ccgaacatgt acaaccttgt gctggccatg 960ttgtggcgtc accctgaaaa tgttgaactt gataaagttc aagtggttca ttactgtgct 1020gctgggtcta agccttggag gttcactggg aagtaactgc aggtcatcaa ctactccaag 1080ctccgtatat gggagtttgt ggagtacagc aagatgatat acttggacgg agacattgag 1140gtatatgaga acatagacca cctatttgac ctacctgatg gtaactttta cgctgtgatg 1200gattgtttct gcgagaagac atggagtcac acccctcagt acaaggtggg ttactgccag 1260caatgcccgg agaaggtgcg gtggcccacc gaattgggtc agcccccttc tctttacttc 1320aacgctggca tgttcgtgtt cgaacccaac atcgccacct atcatgacct attgaaaacg 1380gtgcaagtca ccactcccac ctcgttcgct gaacaagatt tcttgaacat gtacttcaag 1440gacatttaca agccaatccc tttaaattac aatcttgtcc tcgccatgct gtggcgccac 1500ccggaaaacg ttaaattaga ccaagtcaag gttgttcact attgcgcagc ggggtccaag 1560ccatggagat atacggggaa gtagc 1585

Claims

1-21. (canceled)

22. An isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having galactinol synthase activity, wherein the polypeptide has an amino acid sequence of at least 95% identity when compared to SEQ ID NO: 6, or (b) a fragment of the isolated polynucleotide in (a) that functions to co-suppress endogenous nucleic acid sequences encoding polypeptides having galactinol synthase activity.

23. The polynucleotide of claim 22, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:6.

24. The polynucleotide of claim 22, wherein the nucleotide sequence comprises SEQ ID NO:5.

25. A vector comprising the polynucleotide of claim 22.

26. A recombinant DNA construct comprising the polynucleotide of claim 22 operably linked to at least one regulatory sequence.

27. A method for transforming a cell, said method comprising transforming a cell with the polynucleotide of claim 22.

28. A cell comprising the recombinant DNA construct of claim 26.

29. A method for producing a plant, said method comprising transforming a plant cell with the polynucleotide of claim 22 and regenerating a plant from the transformed plant cell.

30. A plant comprising the recombinant DNA construct of claim 26.

31. A seed comprising the recombinant DNA construct of claim 26.