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WO1999036551A1 - Homologues de phosphoglucomutase vegetale - Google Patents

Homologues de phosphoglucomutase vegetale Download PDF

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Publication number
WO1999036551A1
WO1999036551A1 PCT/US1999/000883 US9900883W WO9936551A1 WO 1999036551 A1 WO1999036551 A1 WO 1999036551A1 US 9900883 W US9900883 W US 9900883W WO 9936551 A1 WO9936551 A1 WO 9936551A1
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Prior art keywords
nucleic acid
acid fragment
isolated nucleic
substantial portion
seq
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PCT/US1999/000883
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English (en)
Inventor
William D. Hitz
Stephen M. Allen
Florence Kirsch
J. Antoni Rafalski
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E.I. Du Pont De Nemours And Company
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Priority to AU23214/99A priority Critical patent/AU2321499A/en
Priority to BR9907164-9A priority patent/BR9907164A/pt
Publication of WO1999036551A1 publication Critical patent/WO1999036551A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding phosphoglucomutase proteins in plants and seeds. BACKGROUND OF THE INVENTION
  • Starch synthesis occurs in the chloroplast while soluble carbohydrate (i.e., sucrose) synthesis occurs in the cytosol.
  • These biosynthetic pathways are competing processes because excess triose phosphate can be used for either starch synthesis in the chloroplast or sucrose synthesis in the cytosol.
  • These pathways have many common steps; however, the enzymes that catalyze similar steps are unique to each compartment.
  • These enzymes are isozymes: different forms of the enzymes that catalyze the same reaction. For example, the plastidic and cytosolic forms of phosphoglucomutase both catalyze the conversion of glucose-6-phosphate to glucose 1 -phosphate in different subcellular locations.
  • soybean seed dry weight is protein and 20% is extractable storage oil. These constitute the economically valuable products of the soybean crop.
  • 40% of seed weight about 10% is soluble carbohydrate.
  • the soluble carbohydrate portion contributes little to the economic value of soybean seeds and the main component of the soluble carbohydrates, the raffinosaccharides, are deleterious to both processing and to the food value of soybean meal in monogastric animals (Coon, C. N. et al. Proceedings Soybean Utilization Alternatives, University of Minnesota, (1988) 203-211).
  • the size of the starch and soluble carbohydrate pools present in soybean seeds during the maturation phase indicates that a large portion of the glucose which is converted to raffinose and stachyose during soybean seed maturation comes from the breakdown of a starch pool which was produced slowly during the primary growth phase. Elimination of this transient starch pool may be a strategy for diverting carbon away from the soluble carbohydrate components of dry soybean seeds (sucrose, raffinose and stachyose) and into the more economically desirable components such as oil and protein.
  • the instant invention relates to an isolated nucleic acid fragment encoding a plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein.
  • this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding the plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein.
  • the instant invention relates to a chimeric gene encoding a plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein or to a chimeric gene that comprises an isolated nucleic acid fragment that is complementary to a nucleic acid fragment encoding a plastidic phosphoglucomutase or cytosolic phosphoglucomutase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
  • the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a plastidic phosphoglucomutase or a cytosolic phosphoglucomutase protein operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell.
  • the transformed host cells can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms.
  • the invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
  • An additional embodiment of the instant invention concerns a method of altering the level of expression of a plastidic phosphoglucomutase or a cytosolic phosphoglucomutase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a plastidic phosphoglucomutase or a cytosolic phosphoglucomutase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the a plastidic phosphoglucomutase or a cytosolic phosphoglucomutase in the transformed host cell.
  • An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a plastidic phosphoglucomutase or a cytosolic phosphoglucomutase protein.
  • the invention also relates to soybean lines in which the expression of phosphoglucomutase activity in the plastids of developing seed is decreased due to the transgenic expression of a chimeric gene comprising an isolated nucleic acid fragment encoding the plastid form of a soybean phosphoglucomutase or a chimeric gene that comprises an isolated nucleic acid fragment that is complementary to the nucleic acid fragment encoding the plastid form of a soybean phosphoglucomutase, operably linked to suitable regulatory sequences.
  • Figure 1 depicts starch and soluble sugar metabolism in developing soybean cotyledons.
  • the enzymes abbreviated in the figure are: hexokinase (HK); phosphoglucoisomerase (PGI); UDPglucose pyrophosphorylase (UDPGPP); myo-inositol 1 -phosphate synthase (MI 1-PS); myo-inositol 1 -phosphate phosphatase (MI 1-Pase); sucrose synthase (SS); phosphoglucomutase (PGM); UDP 4'epimerase (EPI); sucrose phosphate synthase (SPS); sucrose 6-phosphatase (S6Pase); ADP-glucose pyrophosphorylase (ADPGPP); galactionol synthase (GAS); raffinose synthase (RS); and stachyose synthase (StS).
  • HK hexokinase
  • PGI phosphoglucoisomerase
  • Glucose originating from starch breakdown can be converted to any of the precursors to raffinose and stachyose through the action of the reversible enzymes of the pathway or by the normal path to sucrose synthesis, SPS-catalyzed formation of sucrose-6-phosphate.
  • FIG. 2 depicts a portion of the output of a Gapped BLASTP search (BLASTP 2.0.3;
  • SEQ ID NO: 1 is the nucleotide sequence comprising the cDNA insert in clone pTC6b encoding a truncated plastid form of a soybean phosphoglucomutase gene.
  • SEQ ID NO:2 is the 5' PCR primer used to amplify that portion of clone pTC6b that encodes a truncated phosphoglucomutase. The sequence of this primer corresponds to nucleotides 494 to 514 of SEQ ID NO:l.
  • SEQ ID NO:3 is the 3' PCR primer used to amplify that portion of clone pTC6b that encodes a truncated phosphoglucomutase. The sequence of this primer corresponds to nucleotides 1017 to 1037 of SEQ ID NO:l.
  • SEQ ID NO:4 is the nucleotide sequence comprising a portion of the cDNA insert in clone pTC15b encoding an entire plastid form of a soybean phosphoglucomutase.
  • SEQ ID NO: 5 is the deduced amino acid sequence of a plastid form of a soybean phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO:4.
  • SEQ ID NO:6 is the 5' PCR primer used to amplify a portion of the cDNA insert in clone pTC15b for expression of the soybean phosphoglucomutase in E. coli.
  • the sequence of this primer corresponds to nucleotides 332 to 351 of SEQ ID NO:4.
  • SEQ ID NO:7 is the 3' PCR primer used to amplify a portion of the cDNA insert in clone pTC15b for expression of the soybean phosphoglucomutase in E. coli.
  • sequence of this primer is derived from the nucleotide sequence of the 3' portion of the insert in clone pTC15b located beyond (3') the apparent poly A tail of the phosphoglucomutase gene.
  • SEQ ID NO:8 is the 5' PCR primer used to amplify a portion of clone pTC15b for expression of a truncated form of the soybean phosphoglucomutase in plants.
  • the sequence of this primer corresponds to nucleotides 1 to 20 of SEQ ID NO:4.
  • SEQ ID NO:9 is the 3' PCR primer used to amplify that portion of clone pTC15b for expression of a truncated form of the soybean phosphoglucomutase in plants.
  • the sequence of this primer corresponds to nucleotides 1370 to 1390 of SEQ ID NO:4.
  • SEQ ID NO: 10 is the nucleotide sequence of a 0.7 kb Arabidopsis cDNA clone having
  • SEQ ID NO:l 1 is the amino acid sequence encoding a plastidic phosphoglucomutase , from Spinacia oleracea L. having EMBL Accession No. X75898.
  • SEQ ID NO: 12 is the nucleotide sequence of a contig assembled from the cDNA inserts in clones cillc.pk001.il 4 and pOOlO.cbpcp ⁇ lr encoding a portion of a corn plastidic phosphoglucomutase.
  • SEQ ID NO: 13 is the deduced amino acid sequence of a plastidic phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO: 12.
  • SEQ ID NO: 14 is the nucleotide sequence comprising a portion of the cDNA insert in clone rl0n.pk0032.e9 encoding a portion of a rice plastidic phosphoglucomutase.
  • SEQ ID NO: 15 is the deduced amino acid sequence of a plastidic phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO: 14.
  • SEQ ID NO: 16 is the nucleotide sequence comprising a portion of the cDNA insert in clone rlm3n.pk002.p2 encoding a portion of a rice cytosolic phosphoglucomutase.
  • SEQ ID NO: 17 is the deduced amino acid sequence of a cytosolic phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO: 16.
  • SEQ ID NO: 18 is the nucleotide sequence comprising a portion of the cDNA insert in clone sgs4c.pk005.h21 encoding a portion of a soybean cytosolic phosphoglucomutase.
  • SEQ ID NO: 19 is the deduced amino acid sequence of a cytosolic phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO: 18.
  • SEQ ID NO:20 is the nucleotide sequence comprising a portion of the cDNA insert in clone wleln.pk0108.al2 encoding a portion of a wheat cytosolic phosphoglucomutase.
  • SEQ ID NO:21 is the deduced amino acid sequence of a cytosolic phosphoglucomutase derived from the nucleotide sequence of SEQ ID NO:20.
  • an "isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double- stranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
  • tig refers to an assemblage of overlapping nucleic acid sequences to form one contiguous nucleotide sequence. For example, several DNA sequences can be compared and aligned to identify common or overlapping regions. The individual sequences can then be assembled into a single contiguous nucleotide sequence.
  • 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 protein encoded by the DNA 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 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 alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences. 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 nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed.
  • alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein are well known in the art.
  • 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.
  • 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 protein molecule would also not be expected to alter the activity of the protein.
  • 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.
  • the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the sequences exemplified herein.
  • Substantially similar nucleic acid fragments of the instant invention may be identified by the degree of similarity of their encoded polypeptides to the polypeptides disclosed herein as determined by the Hein algorithm (Hein, J. J. (1990) Methods in Enzymology
  • nucleic acid fragments encode polypeptides that are 95% ; identical to the polypeptides encoded by the nucleic acid fragments reported herein.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov BLAST ).
  • a sequence often or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • gene specific oligonucleotide probes comprising 20-30 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).
  • short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
  • the instant specification teaches partial or complete amino acid and nucleotide sequences encoding 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.
  • 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 that encodes all or a substantial portion of the amino acid sequence encoding the plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins as set forth in SEQ ID NO: 1
  • “Synthetic genes” 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 gene segments which are then enzymatically assembled to construct the entire gene. "Chemically synthesized", as related to a sequence ) of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA 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 genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.
  • 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.
  • Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature.
  • a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • 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, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • Coding sequence refers to a DNA 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.
  • Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • 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.
  • an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
  • 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 , gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 75: 1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • the "translation leader sequence” refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence.
  • the translation leader sequence is present in the fully processed mR A upstream of the translation start sequence.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G.D. (1995) Molecular Biotechnology 3:225).
  • the "3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
  • the primary transcript 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.
  • RNA essential RNA
  • cDNA double-stranded DNA that is complementary to and derived from mRNA.
  • Sense RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell.
  • Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense
  • RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • the term "operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • 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.
  • expression 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).
  • Antisense inhibition By which expression of specific genes is decreased or eliminated may from time to time be referred to herein as “gene silencing”.
  • altered levels 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.
  • “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
  • Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
  • chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
  • Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
  • a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.
  • a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
  • an endoplasmic reticulum retention signal may be added.
  • 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. 153:211) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Pat. No. 4,945,050, incorporated herein by reference).
  • a nucleic acid fragment encoding the plastid form of a soybean phosphoglucomutase has been isolated.
  • the identity of polypeptide encoded by this nucleic acid fragment was confirmed by searching public databases for similar nucleic acid and protein coding sequences. Results of this search indicate that the instant nucleic acid fragment encodes a soybean homolog of a spinach phosphoglucomutase enzyme. Further confirmation of the identity of the encoded polypeptide was obtained from experiments demonstrating the expected enzymatic functionality of the encoded polypeptide when expressed in E.
  • Example 2 See Example 2 and the ability of a portion of the instant nucleic acid fragment to mediate co- suppression of endogenous phosphoglucomutase activity in soybean embryos resulting in the expected alteration of phenotype (Examples 3 and 4).
  • Nucleic acid fragments encoding at least a portion of several other phosphoglucomutase proteins 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. Table 1 lists the proteins that are described herein, and the designation of the cDNA clones that comprise the nucleic acid fragments encoding these proteins.
  • the nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous phosphoglucomutases 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).
  • 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).
  • genes encoding other plastid phosphoglucomutase or cytosolic phosphoglucomutase proteins could be isolated directly by using all or a portion of the instant nucleic acid fragment 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 sequence can be designed and synthesized by methods known in the art (Maniatis). Moreover, the 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, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
  • primers can be designed and used to amplify a part or all of the instant sequence.
  • 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.
  • two short segments of the instant nucleic acid fragment 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 poly adeny lie acid tracts to the 3' end of the mRNA precursor encoding plant genes.
  • the second primer sequence may be based upon sequences derived from the cloning vector.
  • the skilled artisan can follow the RACE protocol (Frohman et al., (1988) PNAS USA 55:8998) 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) PNAS USA 86:5613; Loh et al., (1989) Science 243:211). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs (Frohman, M. A. and Martin, G. R., (1989) Techniques 7:165).
  • RACE protocol Frohman et al., (1988) PNAS USA 55:8998
  • 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, R. A. (1984) Adv. Immunol. 36: 1; Maniatis).
  • the nucleic acid fragment of the instant invention may be used to create transgenic plants in which the disclosed plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins 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 the plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins in those cells. Alteration of the level of phosphoglucomutase in cells affects the phenotypic expression of the trait or traits that are controlled, either directly or indirectly, by the level of phosphoglucomutase activity in those cells and the plants and seeds that these cells comprise.
  • Overexpression of the plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins of the instant invention may be accomplished by first constructing a chimeric gene 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 chimeric gene may comprise promoter sequences and translation leader sequences derived from the same gene. 3' Non-coding sequences encoding transcription termination signals may also be provided.
  • the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.
  • Plasmid vectors comprising the instant chimeric gene can then 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 chimeric gene. 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 275:78-86), 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.
  • the chimeric gene described above may be further supplemented by altering the coding sequences to encode a phosphoglucomutase with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K. (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals
  • a chimeric gene designed for co-suppression of the instant phosphoglucomutase proteins can be constructed by linking a gene or a gene fragment encoding the plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins to a plant promoter sequence.
  • a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or a gene fragment in reverse orientation to a plant promoter sequence. Either the co-suppression or antisense chimeric gene could be introduced into plants via transformation wherein expression of the corresponding endogenous gene is reduced or eliminated.
  • a chimeric gene designed for co-suppression of endogenous phosphoglucomutase in soybean cells was constructed (see Example 3). Expression of this chimeric gene in transformed soybean embryos resulted in significant decreases in starch and glucose levels compared to controls.
  • One skilled in the art expects that fertile, seeds from transgenic plants produced from the transformed embroyonic tissue would exhibit the same decreased-starch phenotype.
  • the instant plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies these proteins by methods well known to those skilled in the art.
  • the antibodies are useful for detecting the plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins in situ in cells or in vitro in cell extracts.
  • Preferred heterologous host cells for production of the instant plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins 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 chimeric gene for production of the instant phosphoglucomutases. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the instant phosphoglucomutases.
  • An example of a vector for high level expression of the instant plastidic phosphoglucomutase or cytosolic phosphoglucomutase proteins in a bacterial host is provided (Example 2).
  • nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes.
  • the instant nucleic acid fragment may be used as a restriction fragment length polymorphism (RFLP) marker.
  • RFLP restriction fragment length polymorphism
  • Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragment of the instant invention.
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at., (1987) Genomics 7:174-181) in order to construct a genetic map.
  • nucleic acid fragment 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, D. et al., (1980) Am. J. Hum. Genet.32:3 ⁇ 4-33 ⁇ ). The production and use of plant gene-derived probes for use in genetic mapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986) Plant Mol. Biol. Reporter 4(l):31-4 ⁇ .
  • Nucleic acid probes derived from the instant nucleic acid sequence may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, J. D., et al., In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
  • nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet. 7:149-154).
  • FISH direct fluorescence in situ hybridization
  • 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, H. H. (1989) J. Lab. Clin. Med. 7740:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics 76:325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 2 7:1077-1080), nucleotide extension reactions (Sokolov, B. P. (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter, M. A.
  • 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 56:9402; Koes et al., (1995) Proc. Natl. Acad. Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:75). The latter approach may be accomplished in two ways.
  • 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 plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein.
  • 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.
  • a plant containing a mutation in the endogenous gene encoding a plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein can be identified and obtained.
  • This mutant plant can then be used to determine or confirm the natural function of the plastidic phosphoglucomutase or cytosolic phosphoglucomutase protein gene product.
  • the nucleic acid fraction was enriched for poly-A + RNA by passing total RNA through an oligo-dT cellulose column and eluting the poly-A + RNA with salt as described by Goodman et al. ((1979) Meth. Enzymol. 68:15-90).
  • cDNA was synthesized from the poly-A + RNA enriched fraction using the cDNA Synthesis System (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer's instructions.
  • the resultant double-stranded DNA was methylated by Eco RI DNA methylase (Promega, Madison, WI) treated with T4 DNA polymerase (Life Technologies Incorporated) to create blunt ends.
  • the treated fragment was then ligated to phosphorylated Eco RI linkers using T4 DNA ligase (Pharmacia, Piscataway, NJ).
  • T4 DNA ligase (Pharmacia, Piscataway, NJ).
  • the double-stranded DNA was digested with Eco RJ, separated from excess linkers by passage through a gel filtration column (Sepharose CL-4B, Pharmacia), and ligated to previously digested lambda ZAPTM vector (Stratagene, La Jolla, CA) according to manufacturer's instructions.
  • Ligated DNA was packaged into phage using the Gigapack packaging extract (Stratagene) according to manufacturer's instructions.
  • the resultant cDNA library was amplified as per Stratagene's instructions and stored at -80°C.
  • the cDNA library was used to infect E. coli XL-1 Blue MRF' cells for a total of 300,000 plaque forming units onto six 150 mm diameter petri dishes. Plaques were visible after 12 hours incubation at 37°C. Plates were stored for 8-12 hours at 4°C. Phage were transferred to nitrocellulose filters (Schleicher & Schuell, Keene, NH) in duplicate and denatured in 0.5 M NaOH, 1.5 M NaCl and neutralized in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl and a final rinse in 0.2 X SSC, 0.2 M Tris-HCl, pH 8.0. Filters were dried under vacuum at 80°C for 1 hour.
  • Filters were prehybridized in 100 n L 5X SSC, 100 ug/ml denatured salmon sperm DNA, 5X Denhardt's solution, 50 mM Tris-HCl, pH 8.0, 5.0% dextran sulfate, 0.1% SDS for 3 hours at 50°C. Filters were hybridized with a radiolabelled probe made from a 0.7 kb Arabidopsis cDNA clone having EMBL Accession No. Z25508 (SEQ ID NO: 10). Sequence analysis of this Arabidopsis clone indicates that it encodes a portion of a homolog of the plastidic phosphoglucomutase from Spinacia oleracea L. (EMBL Accession No.
  • pTC3d and pTC6b Two of the primary plaques, designated pTC3d and pTC6b, respectively, were plaque purified and the cDNA inserts excised according to manufacturer's instructions (Stratagene). Sequence data from pTC6b (SEQ ID NO: 1) revealed that this clone encoded a truncated phospho g lucomutase , gene. A nucleic acid fragment encoding the phosphoglucomutase portion of pTC6b was amplified using primers shown in SEQ ID NO: 2 and SEQ ID NO:3. 5'- ACCATTGGAG GGTTTCCATA T -3' (SEQ ID NO: 2)
  • SEQ ID NO:2 corresponds to bases 494 to 514 of SEQ ID NO: 1 ;
  • SEQ ID NO:3 is complementary to bases 1017 to 1037 of SEQ ID NO:l.
  • the resulting 543 bp PCR product was purified using Amicon 50 microconcentrator by centrifuging for 5 minutes in a microcentrifuge. The membrane was washed twice with 300 ⁇ l of water and the remaining sample was collected by inverting the concentrator and centrifuging. The soybean cDNA library described above was screened with the radiolabelled 543 bp PCR fragment as described above. Approximately 20 positive plaques were observed for each of the six plates.
  • E. coli expression vector pET24d (Novagen, Madison, WI).
  • the phosphoglucomutase coding region was amplified using Vent® DNA polymerase from New England Biolabs (NEB, Beverly, MA) supplemented with an additional 2 ⁇ L of 100 mM magnesium sulfate added to each 100 ⁇ L reaction using the oligonucleotide primers shown in SEQ ID NO: 6 anr SEQ ID NO:7.
  • the 5' primer corresponds to bases 332 to 351 in SEQ ID NO:4 and includes ten additional bases in order to provide a 6 base Ncol restriction site and four bases to enhance restriction digestion.
  • the 3' primer (SEQ ID NO: 7) is derived from the nucleotide sequence of the 3' portion of the insert in clone pTC15b located beyond (3') the apparent poly A tail of the phosphoglucomutase gene and includes additional bases which provide a 6 base BamHI site and three extra bases to enhance restriction digestion.
  • the resulting 2.2 kb PCR product was purified from a 1% low melting agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, precipitated with ethanol, and dried. The fragment was ligated into pGEM®-t vector (Promega, Madison, WI) for 18 hours at 16°C. The ligated DNA was introduced into DH10B electrocompetent cells (Life Technologies Inc.) and transformants were selected on 2xYT agar plates containing 150 ⁇ g ampicillin/ml, 80 ⁇ M X-Gal (BioRad, Richmond, CA), 0.1 mM isopropyl-D-thiogalactopyranoside (IPTG, Sigma Chemical Co., St.
  • Plasmid DNA was purified according to the manufacturer's instructions using Wizard® DNA purification system (Promega). Plasmid DNA was restricted with Ncol and BamHI and the 2.2 kb phosphoglucomutase fragment was separated from the other products of digestion in a 1 % NuSieve® GTG® agarose gel (FMC, Rockland, ME). Buffer and agarose contained 10 ⁇ g ethidium bromide/mL for visualization of the 2.2 kb DNA fragment.
  • the 2.2 kb fragment was purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies, Madison, WI) according to the manufacturer's instructions, precipitated with ethanol, and dried.
  • the 2.2 kb phosphoglucomutase fragment was ligated into a NcoI-BamHI restricted pET24d vector with T4 DNA ligase (NEB) at 16°C for 18 hours.
  • the ligation reaction was introduced into BL21(DE3) cells (Novagen) and transformants were selected on 2xYT agar plates containing 50 ⁇ g kanamycin/mL.
  • Cells were harvested by centrifugation after 3 h and re-suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonvl fluoride. A , small amount of 1 mm glass beads were added and the mixture was sonicated three times for about 5 seconds each time with a microprobe sonicator. The mixture was centrifuged and the protein concentration of the supernatant was determined. For assay of phosphoglucomutase activity, the following components were prepared in
  • each 557 ⁇ L assay contained 400 ⁇ L of 100 mM potassium phosphate buffer, 5 ⁇ L of E. coli cell extract, 50 ⁇ L of nicotinamide adenine dinucleotide phosphate, 50 ⁇ L of glucose 1 ,6-diphosphate and 2 ⁇ L of glucose 6-phosphate dehydrogenase.
  • Plasmids containing the truncated phosphoglucomutase gene from soybean cDNA sequence under control of the soybean ⁇ -conglycinin promoter were constructed. The construction of vectors was facilitated by the use of plasmid pCW109, described in World Patent Publication No. WO94/11516, incorporated herein by reference.
  • Vector pCW109 contains an 830 base DNA segment which includes the promoter sequences for the -subunit of the soybean seed storage protein ⁇ -conglycinin, a region with multiple restriction sites, and 1080 bases of 3' regulatory sequence from the common bean seed storage protein, phaseolin.
  • Vector pCW109 was modified to contain a Smal site in the multiple cloning region as well. The junction between the ⁇ -conglycinin promoter and the 3' phaseolin sequence was modified to contain a unique Notl site.
  • a plasmid designated pKS18HH was constructed to confer resistance to hygromycin B to either plants or bacteria transformed with the plasmid.
  • the plasmid was constructed using the following genetic elements: 1) plasmid vector pSP72 (Promega) which was modified by removal of the ⁇ -lactamase coding region, 2) the 35S promoter from cauliflower mosaic virus (CaMV)/Hygromycin B Phosphotransferase (HTP) Nopaline synthase 3' regulatory sequence from Agrobacterium tumefaciens, and 3) the T7 promoter and Shine-Delgarno sequence/HPT/T7 terminator sequence.
  • the Hygromycin B phosphotransferase gene from E. coli strain W677 contained a Klebsiella-de ⁇ ved plasmid (pJR225; Gritz, L. and Davies, J. (1983) Gene 25:179-188).
  • the T7 promoter/HPT/T7 terminator cassette for expression of the HPT enzyme in certain strains of E. coli, such as NovaBlue (Novagen) was obtained from a derivative of a p ⁇ T vector.
  • NovaBlue Novagen
  • ⁇ -conglycinin-3' phaseolin cassette containing a unique Notl site in pCW109 was removed by Hindlll digestion, isolated, and ligated into pKS18HH that had also been digested with Hindlll to give the final transformation vector designated pRB20.
  • pRB20 Ten ⁇ g of pRB20 was digested with Notl (N ⁇ B) for 1 hour at 37°C.
  • the digested plasmid was dephosphorylated with alkaline phosphatase (N ⁇ B) for 30 minutes at room temperature.
  • the reaction was deproteinated by extracting once with phenol/chloroform, once with chloroform, ethanol precipitated, washed once with 70% (v/v) ethanol and dried under vacuum.
  • the truncated phosphoglucomutase fragment was prepared by amplification of DNA obtained from clone pTC15b using Vent® polymerase (N ⁇ B) as described in Example 2, using primers shown in SEQ ID NO: 8 (corresponding to bases 1 to 20 in SEQ ID NO:4) and SEQ ID NO:9 (complementary to bases 1370 to 1390 in SEQ ID NO:4).
  • the oligonucleotides presented in SEQ ID NOS:8 and 9 also contain a Notl restriction site and additional ten bases intended to increase the efficiency of digestion by Notl.
  • Notl-digested pRB20 vector was ligated to 1 ⁇ g of the amplified, Notl-digested truncated phosphoglucomutase fragment using T4 DNA ligase (NEB) for 18 hours at 16°C.
  • Electrocompetent DH10B cells were transformed with the ligation reaction and transformants were selected on 2xYT medium containing 150 ⁇ g hygromycin/mL. Eighteen colonies were cultured in 2xYT medium containing hygromycin and plasmid DNA purified as described in Example 2.
  • Soybean embryogenic suspension cultures were maintained in 35 mL liquid media, SB55 or SBP6 (see Table 4) on a rotary shaker, 150 rpm, at 28°C with mixed fluorescent and incandescent lights on a 16:8 hour day/night schedule. Cultures were subcultured every four weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid medium.
  • Soybean embryogenic suspension cultures were transformed with pRB20-phospho- glucomutasel by the method of particle gun bombardment (Kline et al. (1987) Nature (London) 327:10).
  • a DuPont Biolistic PDS 1000/HE instrument (helium retrofit) was used for these transformations.
  • Approximately 300-400 mg of a four week old suspension culture was placed in an empty 60x15 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 was set at 1000 psi and the chamber is evacuated to a vacuum of 28 inches of mercury. The tissue was placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue , was placed back into liquid and cultured as described above.
  • Transformed embryogenic clusters were removed from liquid culture and placed on a solid agar media (SB 103) containing no hormones or antibiotics. Embryos were cultured for four weeks at 26°C with mixed fluorescent and incandescent lights on a 16:8 hour day/night schedule. During this period, individual embryos were removed from the clusters and the starch contents were assayed.
  • SB 103 solid agar media
  • the somatic soybean embryos described above are the first differentiated tissues in the process of producing fertile, transgenic soybean plants by the embryonic tissue culture bombardment method.
  • the somatic embryos produce the storage products typical of zygotic soybean embryos. These products include the storage lipid with a fatty acid composition very similar to zygotic embryos, the seed storage proteins typical of soybean seeds and at levels that are similar to immature zygotic embryos, and a storage carbohydrate profile similar to zygotic embryos.
  • the next steps in producing fertile plants from the transformed somatic embryos is to transfer individual embryos to media SB 148, which is identical in salts and sugar composition to SB 103 except that the MgCl 2 has been removed and the Gelrite replaced with 0.7% agarose.
  • SB 148 the embryos have gone through a maturation and partial dehydration. During that maturation period much of the transient starch is lost, the raffinosaccharide sugars appear and the embryos begin to yellow.
  • the somatic embryos are further desiccated by transfer to a sterile plate in a high humidity chamber.
  • somatic embryos Five days after transfer the somatic embryos are ready for germination on a media similar to SB71-1 except that the sucrose concentration is reduced to 2% and the Gelrite is replaced with 0.5% agarose (SB71-3). Plantlets that form both roots and shoots are transplanted directly to soil and grown to maturity. Successful production of fertile, transgenic soybean plants from transfo ⁇ ned somatic embryos was disclosed in World Patent Publication No. WO94/11516. In transformation experiments involving alteration of the storage fatty acid profile, a portion of the transformed somatic embryos were sacrificed for analysis as was done in the instant example, and the remainder of embryos from the transformation event were taken through to produce fertile plants.
  • Soybeans (cultivar STS) were planted in late May, 1995. A section of the field chosen for uniformity was observed to determine the initial flowering date for the second and third nodes to flower. Twenty one days after the average flowering date for these two nodes sampling was started and continued at 2 to 4 day intervals until seed drydown. At about one hour after sunrise, 40 to 50 pods were taken from nodes 3 through 5 of plants along approximately 4 feet of row. The seeds were removed from the pod, counted and weighed in bulk to obtain the average fresh weight. The seeds were crushed, placed in a tarred 50 mL plastic screw top tube and frozen rapidly. The frozen seeds were lyophilized overnight and re-weighed to obtain the average dry weight per seed and the initial water content.
  • Detector response factors for sucrose, raffinose and stachyose relative to trehalose were determined in standard mixtures and were used to calculate the content of those sugars in the samples using the trehalose internal standard added in the extraction step. The results were converted to amount of each sugar per , developing seed using the average seed dry weight.
  • the seed residue pellets remaining from the soluble sugar determination were extracted twice more with 3 mLs each of 80% (v/v) methanol to remove any residual glucose.
  • the solvent residue was removed at 50°C under a nitrogen stream, then 2 mL of water was added and the samples were heated to 80°C for 30 min.
  • Two hundred ⁇ L of 1 M potassium phosphate buffer was added, the suspension was allowed to cool and 3 ⁇ L of amyloglucosidase (Sigma) was added.
  • the suspension was incubated at 50°C for 2 hr to digest the starch prior to centrifugation.
  • Glucose released from starch was determined by assay of 3 ⁇ L of the supernatant in 0.5 ml of Sigma HK glucose reagent. Results are expressed as mg of glucose released from starch on a per seed basis again using the averaged seed dry weight for conversion. The results are summarized in Table 6.
  • the starch content increased until at least day 38 after pollination while the seed dry weight increased until approximately day 48. While the seed starch content in the average population began to decline after day 38, on average, 3.9 mg of starch remained in each seed at the time maximum seed dry weight was reached. After maximum seed dry weight was attained, the total soluble carbohydrate content continued to increase while the amount of starch per seed continued to decline. After seed dry weight reached its maximum the total , soluble carbohydrate content increased by an average of 4.2 mg per seed while the starch content decreased by 3.6 mg per seed. Since during this time there was no net import of dry matter, it is possible that the breakdown of starch contributed to the increase in soluble carbohydrate content. At a minimum then the starch breakdown could contribute 31 % of the total soluble carbohydrate present in the seed.
  • EXAMPLE 5 Isolation of Additional Nucleic Acid Fragments Encoding Plant Phosphoglucomutase Homologs Composition ofcDNA Libraries; Isolation and Sequencing ofcDNA Clones cDNA libraries representing mRNAs from various corn, rice, soybean and wheat tissues were prepared. The characteristics of the libraries are described below.
  • cDN A libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAPTM XR libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells.
  • ESTs encoding phosphoglucomutase proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol.
  • a contig is an assemblage of overlapping nucleic acid sequences to form one contiguous nucleotide sequence.
  • the individual sequences were assembled into a contiguous nucleotide sequence encoding a unique corn plastidic phosphoglucomutase protein.
  • the BLAST results for the corn contig the rice EST are shown in Table 8:
  • the sequence of the corn contig composed of clones cillc.pk001.il 4 and pOOlO.cbpcp ⁇ lr is shown in SEQ ID NO:12; the deduced amino acid sequence of this contig is shown in SEQ ID NO: 13.
  • the amino acid sequence set forth in SEQ ID NO: 13 is 46.4% similar to the Spinacia oleracea sequence.
  • the sequence of a portion of the cDNA insert from clone rl0n.pk0032.e9 is shown in SEQ ID NO: 14; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 15.
  • the amino acid sequence set forth in SEQ ID NO: 15 is 43.9% similar to the Spinacia oleracea sequence.
  • the instant corn and rice amino acid sequences share approximately 70% similarity to the soybean sequence set forth in SEQ ID NO:5: the corn sequence is 68.3% similar, and the rice sequence is 71.2% similar to the instant soybean sequence. Sequence alignments and similarities, and BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of plastidic phosphoglucomutase proteins. These sequences represent the first rice and corn sequences encoding a plastidic phosphoglucomutase. The corn, rice and soybean plastidic phosphoglucomutases share significant sequence similarity with each other at the amino acid level, and are clearly distinguishable from the Spinacia oleracea phosphoglucomutase. Characterization ofcDNA Clones Encoding Cytosolic Phosphoglucomutase
  • the BLASTX search using the EST sequence from clone sgs4c.pk005.h21 revealed similarity of the protein encoded by the cDNA to cytosolic phosphoglucomutase from Zea mays (NCBI Identifier No. gi 3294467).
  • the BLAST results for each of these ESTs are shown in Table 9:
  • the sequence of a portion of the cDNA insert from clone rlm3n.pk002.p2 is shown in SEQ ID NO: 16; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 17.
  • the sequence of a portion of the cDNA insert from clone sgs4c.pk005.h21 is shown in SEQ ID NO:18; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:19.
  • the sequence of a portion of the cDNA insert from clone wleln.pk0108.al2 is shown in SEQ ID NO:20; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:21.
  • BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of cytosolic phosphoglucomutase. These sequences represent the first rice, soybean and wheat sequences encoding a cytosolic phosphoglucomutase.
  • a chimeric gene comprising a cDNA encoding phosphoglucomutase protein in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed.
  • the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below.
  • Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110-2209), and bears accession number ATCC 97366.
  • the DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega).
  • Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 BlueTM; Stratagene).
  • Bacterial transformants can be screened by , restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM DNA Sequencing Kit; U.S. Biochemical).
  • the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding phosphoglucomutase protein, and the 10 kD zein 3' region.
  • the chimeric gene described above can then be introduced into corn cells by the following procedure.
  • Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132.
  • the embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long.
  • the embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C.
  • Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
  • the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
  • the plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker.
  • This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT).
  • PAT phosphinothricin acetyl transferase
  • the enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin.
  • the pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al.
  • the particle bombardment method (Klein et al., (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells.
  • gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
  • Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
  • Calcium chloride 50 ⁇ L of a 2.5 M solution
  • spermidine free base (20 ⁇ L of a 1.0 M solution) are added to the particles.
  • the suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 ⁇ L of ethanol. An aliquot (5 ⁇ L) of the DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs).
  • the particles are then accelerated into the corn tissue with a BiolisticTM PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
  • a BiolisticTM PDS-1000/He Bio-Rad Instruments, Hercules CA
  • the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium.
  • the tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter.
  • the petri dish containing the tissue can be placed in the chamber of the PDS-1000 He approximately 8 cm from the stopping screen.
  • the air in the chamber is then evacuated to a vacuum of 28 inches of Hg.
  • the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
  • tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., (1990) Bio/Technology 5:833-839).

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Abstract

L'invention concerne un fragment d'acide nucléique isolé codant pour une phosphoglucomutase. L'invention concerne également la construction d'un gène chimérique codant pour la totalité ou une portion d'une phosphoglucomutase, dans une direction sens ou antisens. L'expression du gène chimérique entraîne la production de taux modifiés de phosphoglucomutase dans une cellule hôte transformée. L'invention concerne en outre des plantes et des graines présentant des taux d'amidons réduits résultant d'une expression réduite du gène codant pour une phosphoglucomutase.
PCT/US1999/000883 1998-01-15 1999-01-13 Homologues de phosphoglucomutase vegetale WO1999036551A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU23214/99A AU2321499A (en) 1998-01-15 1999-01-13 Plant phosphoglucomutase homologs
BR9907164-9A BR9907164A (pt) 1998-01-15 1999-01-13 Fragmento de ácido nucléico isolado, gene quimérico. célula hospedeira, planta transformada, polipeptìdeos, método de alteração do nìvel de expressão de uma protéina, método de obtenção de um fragmento de ácido nucléico, método para diminuição da atividade de fosfoglicomutase e da quantidade de amido em uma semente de soja em desenvolvimento, semente de planta e produto

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7153298P 1998-01-15 1998-01-15
US60/071,532 1998-01-15
US11086698P 1998-12-04 1998-12-04
US60/110,866 1998-12-04

Publications (1)

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WO1999036551A1 true WO1999036551A1 (fr) 1999-07-22

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AU (1) AU2321499A (fr)
BR (1) BR9907164A (fr)
WO (1) WO1999036551A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001075128A3 (fr) * 2000-03-31 2002-04-04 Inst Pflanzengenetik & Kultur Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures
EP1174510A3 (fr) * 2000-07-17 2002-04-24 E.I. du Pont de Nemours and Company Gènes de phosphoglucomutases issus de plastides
US7323560B2 (en) 2000-07-17 2008-01-29 E.I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
WO2009143398A1 (fr) * 2008-05-23 2009-11-26 E. I. Du Pont De Nemours And Company Gènes dgat issus de yarrowia lipolytica combinés à la régulation à la baisse d'une phosphoglucomutase plastidique en vue d'obtenir une production accrue de lipides de stockage de semences et des profils d'acides gras modifiés dans des plantes à graines oléagineuses

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WO1993002196A1 (fr) * 1991-07-24 1993-02-04 E.I. Du Pont De Nemours And Company Sequences de nucleotides de synthase de galactinol de zucchini et de soja
WO1994024309A1 (fr) * 1993-04-16 1994-10-27 Medical Research Council Polymorphismes dans la phosphoglucomutase-1 humaine
WO1994028149A1 (fr) * 1993-05-28 1994-12-08 Monsanto Company Procede d'amelioration de la qualite de pommes de terre stockees
WO1996012812A1 (fr) * 1994-10-21 1996-05-02 Danisco A/S Procede de reduction de la teneur en sucre d'un organisme
WO1998001574A1 (fr) * 1996-07-09 1998-01-15 Unilever Plc Procede pour augmenter la teneur en sucrose des plantes
WO1998022604A1 (fr) * 1996-11-20 1998-05-28 Pioneer Hi-Bred International, Inc. Procedes de production de graine a haute teneur en huile par modification des teneurs en amidon

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MICHALOWSKI, C.B., ET AL.: "Mesembryanthemum crystallinum phosphoglucomutase (PGM) mRNA, complete cds.", EMBL ACCESSION NO. U84888, 8 February 1997 (1997-02-08), XP002100926 *
MUELLER-ROEBER B ET AL: "APPROACHES TO INFLUENCE STARCH QUANTITY AND STARCH QUALITY IN TRANSGENIC PLANTS", PLANT CELL AND ENVIRONMENT, vol. 17, no. 5, 1 January 1994 (1994-01-01), pages 601 - 613, XP002005943 *
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001075128A3 (fr) * 2000-03-31 2002-04-04 Inst Pflanzengenetik & Kultur Procede de production de legumineuses a teneur en proteines et duree de remplissage du grain superieures
EP1174510A3 (fr) * 2000-07-17 2002-04-24 E.I. du Pont de Nemours and Company Gènes de phosphoglucomutases issus de plastides
US7250557B2 (en) 2000-07-17 2007-07-31 E. I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
US7323560B2 (en) 2000-07-17 2008-01-29 E.I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
US7915486B2 (en) 2000-07-17 2011-03-29 E. I. Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
US8143479B2 (en) 2000-07-17 2012-03-27 E I Du Pont De Nemours And Company Plastidic phosphoglucomutase genes
WO2009143398A1 (fr) * 2008-05-23 2009-11-26 E. I. Du Pont De Nemours And Company Gènes dgat issus de yarrowia lipolytica combinés à la régulation à la baisse d'une phosphoglucomutase plastidique en vue d'obtenir une production accrue de lipides de stockage de semences et des profils d'acides gras modifiés dans des plantes à graines oléagineuses
US8143476B2 (en) 2008-05-23 2012-03-27 E I Du Pont De Nemours And Company DGAT genes from Yarrowia lipolytica combined with plastidic phosphoglucomutase down regulation for increased seed storage lipid production and altered fatty acid profiles in oilseed plants
US8829273B2 (en) 2008-05-23 2014-09-09 E.I. Du Pont De Nemours And Company DGAT genes from Yarrowia lipolytica for increased seed storage lipid production and altered fatty acid profiles in soybean
US9574207B2 (en) 2008-05-23 2017-02-21 E I Du Pont De Nemours And Company DGAT genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants
US10087456B2 (en) 2008-05-23 2018-10-02 E I Du Pont De Nemours And Company DGAT genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

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BR9907164A (pt) 2000-11-14

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