WO2000061735A9 - Modification des reserves des plantes - Google Patents

Modification des reserves des plantes

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Publication number
WO2000061735A9
WO2000061735A9 PCT/US2000/009192 US0009192W WO0061735A9 WO 2000061735 A9 WO2000061735 A9 WO 2000061735A9 US 0009192 W US0009192 W US 0009192W WO 0061735 A9 WO0061735 A9 WO 0061735A9
Authority
WO
WIPO (PCT)
Prior art keywords
sse
plant
polypeptide
cell
nucleic acid
Prior art date
Application number
PCT/US2000/009192
Other languages
English (en)
Other versions
WO2000061735A1 (fr
WO2000061735A8 (fr
Inventor
Yun Lin
Lin Sun
Long V Nguyen
Howard M Goodman
Original Assignee
Gen Hospital Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gen Hospital Corp filed Critical Gen Hospital Corp
Publication of WO2000061735A1 publication Critical patent/WO2000061735A1/fr
Publication of WO2000061735A8 publication Critical patent/WO2000061735A8/fr
Publication of WO2000061735A9 publication Critical patent/WO2000061735A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • 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
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to the deposition of plant storage reserve materials (e.g., seed reserve material), the biogenesis of storage organelles, and the production of transgenic plants having altered storage reserve profiles.
  • plant storage reserve materials e.g., seed reserve material
  • the invention features an isolated nucleic acid molecule which includes a sequence encoding an SSE polypeptide.
  • the isolated nucleic acid molecule which includes a sequence encoding a polypeptide that is substantially identical to SSEl (SEQ ID NO:2).
  • the sequence encodes an SSE polypeptide having at least 30% identity with the amino acid sequence shown in Fig. 2B (SEQ ID NO:2).
  • the sequence encodes an SSE polypeptide that, when expressed in a cell of a plant, modifies or alters the production of a food storage reserve material (e.g., protein, lipid, or carbohydrate storage reserve); facilitates the intracellular transport of a storage protein; or facilitates the formation of protein or oil bodies.
  • the nucleic acid molecule is a cDNA molecule.
  • the invention features an isolated nucleic acid molecule which includes a sequence that encodes an SSE polypeptide, wherein the isolated nucleic acid molecule hybridizes specifically to a nucleic acid molecule that includes the cDNA of Fig. 2A (SEQ ID NO:l).
  • the isolated nucleic acid sequence encodes an SSE polypeptide having at least 30% identity with the amino acid sequence shown in Fig. 2B (SEQ ID NO:2).
  • the invention features a transgenic plant (or plant cell, plant tissue, plant organ, or plant component) which includes a recombinant transgene that expresses an SSE polypeptide, wherein the transgene is expressed in the transgenic plant under the control of an expression control region that is functional in a plant cell.
  • the invention further features seeds and cells produced by a transgenic plant which includes such a recombinant transgene.
  • the invention features a sense-oriented expression vector which includes any ofthe aforementioned nucleic acid molecules; the vector being capable of directing expression ofthe SSE polypeptide encoded by the nucleic acid molecule.
  • the invention also includes a cell (e.g., a bacterial or plant cell) or a transgenic plant or transgenic plant component that includes such an expression vector.
  • the invention features an expression vector for producing antisense SSE RNA; a transgenic plant or transgenic plant component including such an antisense vector; and seeds or cells produced by a transgenic plant or transgenic plant component that express the antisense construct.
  • the invention features a substantially pure SSE polypeptide that includes an amino acid sequence having at least 30% identity to the amino acid sequence of Fig. 2B (SEQ ID NO:2).
  • the polypeptide modifies or alters the production of a storage reserve (e.g., a protein or lipid storage reserve); facilitates the intracellular transport of a storage protein or lipid; or facilitates the formation of protein bodies or oil bodies.
  • a storage reserve e.g., a protein or lipid storage reserve
  • facilitates the intracellular transport of a storage protein or lipid or facilitates the formation of protein bodies or oil bodies.
  • the invention features a method of producing an SSE polypeptide, the method includes the steps of: (a) providing a cell transformed with a nucleic acid molecule ofthe invention positioned for expression in the cell; (b) culturing the transformed cell under conditions for expressing the nucleic acid molecule; and (c) recovering the SSE polypeptide. Recombinant SSE polypeptides produced using this method are also included in the invention.
  • the invention features a substantially pure antibody that specifically recognizes and binds to an SSE polypeptide or a portion thereof.
  • the antibody specifically recognizes and binds to a recombinant SSE polypeptide or a portion thereof.
  • the invention features a method of isolating an SSE gene or fragment thereof, the method including the steps of: (a) contacting the nucleic acid molecule of Fig. 2A (SEQ ID NO:l) or a portion thereof with a nucleic acid preparation from a plant cell under hybridization conditions providing detection of nucleic acid sequences having at least 30%> or greater sequence identity to the nucleic acid sequence of Fig. 2A (SEQ ID NO: 1); and (b) isolating the hybridizing nucleic acid sequences.
  • the invention features a method of isolating an SSE gene or fragment thereof, the method including the steps of: (a) providing a sample of plant cell DNA; (b) providing a pair of oligonucleotides having sequence identity to a region ofthe nucleic acid of Fig. 2A (SEQ ID NO: 1); (c) contacting the pair of oligonucleotides with the plant cell DNA under conditions suitable for polymerase chain reaction-mediated DNA amplification; and (d) isolating the amplified SSE gene or fragment thereof.
  • the amplification step is carried out using a sample of cDNA prepared from a plant cell.
  • the pair of oligonucleotides used in the amplification step are based on a sequence encoding an SSE polypeptide, wherein the SSE polypeptide is at least 30% identical to the amino acid sequence of Fig. 2B (SEQ ID NO:2).
  • the invention features a method for modifying or altering the biosynthesis of a storage reserve in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into a plant cell a transgene including DNA encoding an SSE polypeptide having at least 20% identity to the SSEl polypeptide (SEQ ID NO: 2) operably linked to a promoter functional in plant cells to yield a transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from said transformed cells, wherein the SSE polypeptide is expressed in the cells of said transgenic plant or transgenic plant component, thereby modifying or altering the seed storage reserve of said transgenic plant or transgenic plant component.
  • the storage reserve material is a lipid, a storage protein, or a carbohydrate (e.g., a starch).
  • the expressed polypeptide is Pexl ⁇ (SEQ ID NO:6).
  • the storage reserve is a seed or vegetative storage reserve material.
  • the invention features a method for modifying or altering the biosynthesis of a storage reserve in a transgenic plant cell, the method including reducing the level of an SSE polypeptide (or expression of an SSE gene) in a transgenic plant or transgenic plant component.
  • the method for reducing the level ofthe SSE polypeptide includes expressing an antisense SSE nucleic acid sequence in the transgenic plant or transgenic plant component.
  • the method for reducing the level of an SSE polypeptide includes co-suppression of an SSE nucleic acid sequence in the transgenic plant or transgenic plant component.
  • the storage reserve material is a lipid, a storage protein, or a carbohydrate (e.g., a starch).
  • the storage reserve is a seed or vegetative storage reserve material.
  • the invention features a process for modifying storage protein production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying storage protein production in the transgenic plant or transgenic plant component.
  • the transgene encoding the SSE polypeptide is overexpressed.
  • the transgene encoding the SSE polypeptide is constitutively expressed, is inducibly expressed, or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage protein production is increased relative to an untransformed control plant or plant component.
  • the invention also features a process for modifying storage protein production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an antisense coding sequence of an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the antisense coding sequence ofthe SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying storage protein production in the transgenic plant or transgenic plant component.
  • the transgene encoding an antisense coding sequence of an SSE polypeptide is overexpressed.
  • the transgene encoding an antisense coding sequence of an SSE polypeptide is constitutively expressed.
  • the transgene encodes an antisense coding sequence of an SSE polypeptide is inducibly expressed or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage protein production is decreased relative to an untransformed control plant or plant component.
  • the invention features a process for modifying storage lipid production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying storage lipid production in the transgenic plant or transgenic plant component.
  • the transgene encoding the SSE polypeptide is overexpressed.
  • the transgene encoding the SSE polypeptide is constitutively expressed, is inducibly expressed, or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage lipid production is increased relative to an untransformed control plant or plant component.
  • the invention features a process for modifying storage lipid production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an antisense coding sequence of an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the antisense coding sequence of the SSE polypeptide is expressed in the cells of the transgenic plant or transgenic plant component, thereby modifying storage lipid production in the transgenic plant or transgenic plant component.
  • the transgene encoding an antisense coding sequence of an SSE polypeptide is overexpressed.
  • the transgene encoding an antisense coding sequence of an SSE polypeptide is constitutively expressed.
  • the transgene encodes an antisense coding sequence of an SSE polypeptide is inducibly expressed or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage lipid production is decreased relative to an untransformed control plant or plant component.
  • the invention features a process for modifying storage carbohydrate production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying storage carbohydrate production in the transgenic plant or transgenic plant component.
  • the transgene encoding the SSE polypeptide is overexpressed.
  • the transgene encoding the SSE polypeptide is constitutively expressed, is inducibly expressed, or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage carbohydrate production e.g., starch production
  • the invention features a process for modifying storage carbohydrate production in a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an antisense coding sequence of an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the antisense coding sequence ofthe SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying storage carbohydrate production in the transgenic plant or transgenic plant component.
  • the transgene encoding an antisense coding sequence of an SSE polypeptide is constitutively expressed.
  • the transgene encodes an antisense coding sequence of an SSE polypeptide is inducibly expressed or is expressed in a tissue-specific, cell-specific, or organ-specific manner.
  • storage carbohydrate production e.g., starch production
  • the invention features a process for modifying dessication tolerance of a transgenic plant or transgenic plant component, the method including the steps of: (a) introducing into plant cells a transgene encoding an antisense coding sequence of an SSE polypeptide operably linked to a promoter functional in the plant cells to yield transformed plant cells; and (b) regenerating a transgenic plant or transgenic plant component from the transformed plant cells, wherein the antisense coding sequence ofthe SSE polypeptide is expressed in the cells ofthe transgenic plant or transgenic plant component, thereby modifying dessication tolerance ofthe transgenic plant or transgenic plant component.
  • the dessication tolerance ofthe transgenic plant or transgenic plant component is increased relative to an untransformed control plant or plant component.
  • SSE shrunken seed gene or “SSE” gene is meant a gene encoding a polypeptide that governs or regulates protein and oil body biogenesis in a plant cell. SSE genes may be identified and isolated from any plant species, especially agronomically important crop plants, using any ofthe sequences disclosed herein in combination with conventional methods known in the art.
  • polypeptide is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 30%, preferably 50%>, more preferably 80%o, and most preferably 90%o, or even 95%> homology to a reference amino acid sequence (for example, the amino acid sequence shown in Fig. 2B (SEQ ID NO:2) or nucleic acid sequence (for example, the nucleic acid sequences shown in Fig. 2A (SEQ ID NO:l)).
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater.
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater. Sequence identity is typically measured using sequence analysis software
  • Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • substantially pure polypeptide an SSE polypeptide (for example, an SSE polypeptide such as SSEl (SEQ ID NO:2)) that has been separated from components which naturally accompany it.
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%>, and most preferably at least 99%, by weight, an SSE polypeptide.
  • a substantially pure SSE polypeptide may be obtained, for example, by extraction from a natural source (for example, a plant cell); by expression of a recombinant nucleic acid encoding an SSE polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • derived from or “obtained from” is meant isolated from or having the sequence of a naturally-occurring sequence (e.g., a cDNA, genomic DNA, synthetic, or a combination thereof).
  • isolated nucleic acid molecule is meant a DNA molecule that is free ofthe genes which, in the naturally-occurring genome ofthe organism from which the DNA ofthe invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
  • hybridizes specifically is meant that a nucleic acid sequence hybridizes to a DNA sequence at least under low stringency conditions as described herein, and preferably under high stringency conditions, also as described herein.
  • antisense SSE is meant a nucleotide sequence that is complementary to an SSE (or SSE homolog) messenger RNA.
  • an antisense sequence will usually be at least 15 nucleotides, preferably about 15-200 nucleotides, and more preferably 200-2,000 nucleotides in length.
  • the antisense sequence may be complementary to all or a portion of the SSE or SSE homolog mRNA nucleotide sequence (for example, the SSEl gene), and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence binds as well as the length ofthe antisense sequence will vary, depending upon the degree of inhibition desired and the uniqueness ofthe antisense sequence.
  • a transcriptional construct expressing an SSE antisense nucleotide sequence includes, in the direction of transcription, a promoter, the sequence coding for the antisense RNA on the sense strand, and a transcriptional termination region.
  • Antisense SSE sequences may be constructed and expressed according to standard methods, for example, in van der Krol et al., Gene 72:45, 1988; Rodermel et al., Cell 55:673, 1988; Mol et al., FEBS Lett. 268:427, 1990; Weigel and Nilsson, Nature 377: 495, 1995; Cheung et al, Cell 82:383, 1995; and U.S. Pat. No. 5,107,065.
  • transformed cell is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) an SSE polypeptide.
  • positioned for expression is meant that the DNA molecule is positioned adjacent to a DNA sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, for example, an SSE polypeptide, a recombinant protein, or an RNA molecule).
  • reporter gene is meant a gene whose expression may be assayed; such genes include, without limitation, ⁇ -glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), ⁇ - galactosidase, herbicide resistant genes and antibiotic resistance genes.
  • GUS ⁇ -glucuronidase
  • CAT chloramphenicol transacetylase
  • GFP green fluorescent protein
  • ⁇ - galactosidase herbicide resistant genes and antibiotic resistance genes.
  • expression control region is meant any minimal sequence sufficient to direct transcription. Included in the invention are promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers); such elements may be located in the 5' or 3' regions ofthe native gene or engineered into a transgene construct.
  • promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress-, or hormone-inducible elements or chemical inducers); such elements may be located in the 5' or 3' regions ofthe native gene or engineered into a transgene construct.
  • operably linked is meant that a gene and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
  • Plant cell is meant any self-propagating cell bounded by a semi- permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired.
  • Plant cell includes, without limitation, algae, cyanobacteria, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
  • crucifer any plant that is classified within the Cruciferae family.
  • the Cruciferae include many agricultural crops, including, without limitation, rape (for example, Brassica campestris and Brassica napus), broccoli, cabbage, brussel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis.
  • rape for example, Brassica campestris and Brassica napus
  • broccoli cabbage, brussel sprouts, radish, kale, Chinese kale, kohlrabi, cauliflower, turnip, rutabaga, mustard, horseradish, and Arabidopsis.
  • transgene any piece of DNA which is inserted by artifice into a cell, and becomes part ofthe genome ofthe organism which develops from that cell.
  • a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene ofthe organism.
  • transgenic is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part ofthe genome ofthe organism which develops from that cell.
  • the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into the nuclear or plastidic genome.
  • a transgenic plant according to the invention may contain one or more acquired resistance genes.
  • detectably-labelled any direct or indirect means for marking and identifying the presence of a molecule, for example, an ohgonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule or a fragment thereof.
  • Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (for example, with an isotope such as 32 P or 35 S) and nonradioactive labelling (for example, chemiluminescent labelling, for example, fiuorescein labelling).
  • purified antibody is meant antibody which is at least 60%>, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably 90%), and most preferably at least 99%>, by weight, antibody, for example, an acquired resistance polypeptide-specific antibody.
  • a purified SSE antibody may be obtained, for example, by affinity chromatography using a recombinantly-produced acquired resistance polypeptide and standard techniques.
  • telomere binding protein By “specifically binds” is meant an antibody which recognizes and binds an SSE protein but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes an SSE protein such as S S E 1.
  • the invention provides a number of important advances and advantages for engineering plant storage reserves, including seed and vegetative reserve storage material.
  • the invention facilitates an effective and economical means for producing plants having increased nutritional value.
  • the invention further provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals.
  • the invention contributes to the production of high quality and high yield agricultural products: for example, fruits, ornamentals, vegetables, legumes, cereals and field crops.
  • the invention is also useful for providing nucleic acid and amino acid sequences of an SSE gene that facilitates the isolation and identification of SSE genes from any plant species.
  • Figure 1 is a panel of photographs showing the abnormal storage deposition and the shrunken phenotype of ssel seeds.
  • Figures 1 A and IB are transmission electron micrographs of a representative cell from the wild-type cotyledon and hypocotyl, respectively.
  • Figures 1C and ID are transmission electron micrographs of a representative cell from the ssel cotyledon and hypocotyl, respectively.
  • Wild-type cells are filled with numerous oil bodies (OB) and a few large protein bodies (PB).
  • ssel cells contained few oil bodies and additional structures such as starch granules (St), vacuoles (Vc), stacks of membranes (M), and vesicles (Vs).
  • Figures IE and IF show photographs of wild-type C24 and ssel seeds, respectively.
  • the magnification bar found in Figs. 1A-1D is 3.1 ⁇ M.
  • ssel seeds were imbibed in water for 20 minutes before processing. Seeds were cut into halves and fixed in 2.5% glutaraldehyde/0.1 M cacodylate buffer (pH 7.2), post-fixed in 1% osmium tetroxide, dehydrated in an ethanol series, and embedded in Spurr's resin. Thin sections were then stained with uranyl acetate and observed under a transmission electron microscope.
  • Figure 2A shows the cDNA sequence (SEQ ID NO: 1) of SSEl .
  • the ATG start codon and TGA stop codon ofthe SSEl gene are located at positions 122 and 1223, respectively.
  • Figure 2B shows the predicted amino acid sequence of SSEl (SEQ ID NO:2) encoded by the cDNA shown in Fig. 2A. Hydrophobic (single line) and hydrophilic (double line) regions ofthe polypeptide are underlined.
  • Figure 3 A is a PCR diagram showing that primers A and B amplify an ⁇ 0.9-kb fragment from the wild-type SSEl allele, and primers C and B amplify an ⁇ 1.6-kb fragment from the T-DNA interrupted ssel allele.
  • Figure 3B shows the single seed PCR results of round (R) and shrunken (S) seeds in a F 2 population derived from a backcross between a T2 and a wild-type plant.
  • Figure 4A shows the amino acid sequence alignment of SSEl (SEQ ID NO:2) and Pexl ⁇ p (SEQ ID NO:6). Dots indicate gaps. Identical residues are boxed. Hydrophobic (single line) and hydrophilic (double line) regions for both proteins are underlined (Kyte and Doolittle, J. Mol. Biol. 157:105, 1982). The predicted glycosylation site of SSEl is indicated with an asterisk.
  • Figure 4B shows the phenotype of seeds obtained from ssel plants expressing the SSEl transgene.
  • Figure 4C shows the SSEl complementation of pex 16 mutants pex 16-1 and P16KO-8A (Eitzen et al., J. Cell Biol. 137:1265, 1997) for growth on oleic acid as sole carbon source.
  • SSEl cDNA was cloned into the EcoRI site of a Y. lipolytica shuttle vector pTc3 between the promoter and the terminator regions of Y. lipolytica thiolase gene.
  • Ura + transformants of pex 16-1 and P16KO-8A were obtained as described by Eitzen et al. (supra).
  • E122 is the wild-type strain.
  • Figure 4D shows SSEl complementation of pexl6-l mutant for the dimorphic transition from yeast to mycelia form.
  • Cells were grown at 30°C in YND liquid medium (Eitzen et al., supra).
  • the SSEl transformant underwent dimorphic transition at a lower frequency than the wild-type strain El 22.
  • Figure 5 shows the results of competitive RT-PCR analyses of SSEl expression profiles.
  • RNA was isolated from flowers before (B), on the day (0), or 1 day after pollination (1); from siliques 3 to 21 days after pollination; from cotyledons of 2-day-old seedlings; and from expanding rosette leaves and roots.
  • An equal amount of competitor cDNA template was included in each reaction.
  • the SSEl target (T)-to-competitor (C) cDNA ratios reflect the relative expression levels ofthe SSEl gene.
  • the ssel mutant was identified in a transferred DNA (T-DNA) transgenic line (T line) that exhibited the shrunken seed phenotype as follows.
  • T-DNA transferred DNA
  • the cDNA of the Arabidopsis prohibitin gene Atphbl (Genbank Accession Number: U66591) in an antisense orientation was inserted into pBI121 (Clontech, LaJolla, CA) between the Sad and BamHI sites to replaced the ⁇ -glucuronidase coding region.
  • This construct was then used to transform Arabidopsis thaliana C24 according to standard methods. Approximately 2%> ofthe C24 transgenic lines, resulting from transformation experiments, showed the shrunken seed phenotype (ssel).
  • Northern blot analysis with an Atphbl cDNA bottom strand probe showed that the Atphbl mRNA level in ssel was similar to that ofthe wild type.
  • T2 seeds on the TI plant (the primary transgenic plant)
  • 90%> ofthe seeds were shrunken and 10%> were normally rounded.
  • the shrunken seeds were not viable, and plants grown from the round seeds produced -90%) shrunken seeds.
  • Propagation ofthe transgenic line by self-pollination for 4 generation (to T5) showed that this pattern of inheritance continued for generations.
  • ssel was observed to behave as a typical single recessive Mendelian gene. This was shown by reciprocally crossing T2 plants derived from round seeds to wild-type C24 plants. All F j seeds resulting from these crosses were round.
  • DNA fragment flanking the T-DNA was isolated by the thermal asymmetric interlaced-polymerase chain reaction according to the methods described by Liu et al. (Plant J. 8: 457-463, 1995) and used as a probe for screening a genomic library.
  • a 17-kb genomic fragment isolated from a ⁇ -FIXII Arabidopsis C24 genomic library was then used as a probe to screen an Arabidopsis seedling cDNA library which was prepared according to the methods described by Minet et al. (Plant J. 2:417, 1992).
  • Two SSEl cDNA clones were subsequently identified, and DNA sequencing of these clones revealed that both have identical 5' ends and both included stop codons.
  • the 3 ' polyadenylation site was determined by 3 ' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) according to standard methods.
  • the SSEl cDNA sequence (SEQ ID NO:l) and its predicted amino acid sequence (SEQ ID NO:2) are shown in Figures 2A and 2B, respectively.
  • SSEl was found to reside within the BAC clones F17K2 and F4I18 (GenBank Accession Numbers: AC003680 and AC004665, respectively).
  • the SSEl protein predicted by the open reading frame was found to differ from the F17K2.22 hypothetical protein due to discrepancies between the predicted and the actual splicing sites.
  • SSEl sequences obtained were then used to design three primers for determining the genotypes of shrunken and round seeds by single seed polymerase chain reaction (PCR) (Fig. 3A). These experiments were performed as follows. DNA was isolated from single embryos after removal ofthe seed coat, which had the same genotype as the parent.
  • the mixture was centrifuged and the supernatant was mixed with 175 ml ethanol to precipitate the DNA.
  • the DNA was dissolved in 20 ml of water and 1 ml of DNA was used in a 20 ml PCR reaction. As shown in Fig 3 A, primer A
  • the SSEl cDNA encodes a predicted protein of 367 amino acids (SEQ ID NO:2; Figs. 2B and 4A). Expression of SSEl cDNA in transgenic ssel plants was found to complement the shrunken seed phenotype (Fig. 4B). ssel plants were complemented with SSEl as follows. The SSEl cDNA was fused with the 35 S promoter (35SP) and the nopaline synthase 3' region (NOS 3 '). The 35SP-SSE1-NOS3 ' cassette was subcloned into the Kpnl site ofthe pLVN19R binary vector to make the pLVN19R-SSEl construct.
  • 35SP 35 S promoter
  • NOS 3 ' nopaline synthase 3' region
  • T3 plants from round seeds were then vacuum infiltrated with Agrobacterium tumefaciens strain GV3101 (Bechtold et al., C. R. Acad. Sci. Paris Life Sci. 316: 1194, 1993) carrying pLV 19R-SSEl.
  • Genotypes of seven methotrexate resistant transgenic plants were determined by PCR and six were found to be homozygous ssel.
  • Four transgenic ssel plants were fertile and produced complemented T2 seeds at 67 to 87%o. Similar to wild type, transgenic ssel seeds expressing SSEl were tolerant of desiccation, and cells were filled with storage proteins and lipids, but lacked starch. As shown in Fig.
  • the SSEl sequence showed similarity to Pexl ⁇ p, a membrane associated protein required for the assembly and proliferation of peroxisomes (Eitzen et al., supra) and for the trafficking of plasma membrane and cell wall associated proteins (Titorenko et al., Mol. Cell. Biol. 17:5210, 1997), in the yeast Y. lipolytica.
  • Pexl ⁇ p is glycosylated and transiently localized in the endoplasmic reticulum (ER) (Titorenko and Rachubinski, supra).
  • ER endoplasmic reticulum
  • Fig. 4A A predicted glycosylation site was found in SSEl (Fig.
  • SSEl was also found to complement the growth of pexl6 mutants on oleic acid as sole carbon source (Fig. 4C); indicating restoration of peroxisomal function (Eitzen et al., supra).
  • SSEl partially complemented the pexl 6-1 mutant for the dimorphic transition from yeast to the mycelia form (Fig. 4D). Pexl ⁇ p is normally required for mycelia phase specific cell surface protein transport.
  • Peroxisomes are not generally found in dry seeds (Olsen and Harada, Annu. Rev. Plant Physiol. Plant Mol. Biol. 46:123, 1995 and references cited therein; Fig. 1). Protein and oil bodies are the most abundant organelles in mature Arabidopsis seeds and the formation of both is ER-dependent (Mansfield and Briarty, supra;
  • SSEl gene expression was also analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR).
  • RT-PCR competitive reverse transcription-polymerase chain reaction
  • the amount of SSEl mRNA obtained from different tissues and organs was determined as the target-to-competitor cDNA ratio as follows. After deoxyribonuclease treatment, 1 mg RNA was reverse transcribed in a 20 ml reaction, with 0.4 mM ofthe SSEl specific primer FP15R (5'-GGCAATATTCTTCCGTTGC-3'; SEQ ID NO:7). Subsequently, 1 ml ofthe reverse transcription mixture and 5 X 10 "21 mol of competitor cDNA were used in each 20 ml PCR reaction.
  • the competitor cDNA was identical to the SSEl cDNA (designated target cDNA) except for a 95 -bp internal deletion from the EcoRI to the Ncol site.
  • the primers FP7 (5'-AAAAATGGAACTACATTATTCTC-3'; SEQ ID NO:8) and FP14R (5'-ATAAGTAAAACGCTTAACCTHC-3'; SEQ ID NO:9) amplify 814- and 719-bp fragments respectively, from the target and the competitor cDNAs.
  • the ratio ofthe two PCR products reflected the relative amount of SSEl cDNA (or mRNA) in each sample (Siebert and Larrick, Nature 359:557, 1992). The results of these experiments are depicted in Figure 5.
  • SSEl steady state mRNA level in the siliques increased during seed maturation to a maximum in mature 19- and 21 -day-old brown siliques.
  • the level of mRNA was also high in cotyledons of germinating seedlings and flowers, but low in expanding leaves and roots. Glyoxysomes are assembled in germinating seedlings (Olsen and Harada, supra); therefore SSEl is likely to be required in this process.
  • the low expression in expanding leaves, where leaf peroxisomes are formed may be due to low peroxisomes abundance.
  • SSEl may not normally be involved in peroxisome/glyoxysome formation; rather its expression in germinating seedlings may be required for maintenance ofthe remaining oil bodies.
  • the high expression levels in flowers suggests additional functions of SSEl, possibly the formation of oil body like organelles in tapetum and pollen (Huang, supra).
  • any cell or tissue can serve as the nucleic acid source for the molecular cloning of an SSE gene.
  • Isolation of an SSE gene involves the isolation of those DNA sequences which encode a protein exhibiting SSE-associated structures, properties, or activities, for example, the ability to complement an ssel phenotype.
  • the isolation of additional plant SSE coding sequences e.g., those sequences derived from monocots or dicots
  • the SSE sequences described herein may be used, together with conventional screening methods of nucleic acid hybridization screening.
  • all or part ofthe SSEl cDNA may be used as a probe to screen a recombinant plant DNA library for genes having sequence identity to the SSE gene.
  • Hybridizing sequences are detected by plaque or colony hybridization according to the methods described below.
  • SSE-specific ohgonucleotide probes including SSE degenerate ohgonucleotide probes (i.e., a mixture of all possible coding sequences for a given amino acid sequence).
  • SSE degenerate ohgonucleotide probes i.e., a mixture of all possible coding sequences for a given amino acid sequence.
  • These oligonucleotides may be based upon the sequence of either DNA strand and any appropriate portion ofthe SSE sequence (Fig. 2A; SEQ ID NO:l).
  • General methods for designing and preparing such probes are provided, for example, in Ausubel et al., (supra), and Berger and Kimmel, (supra).
  • oligonucleotides are useful for SSE gene isolation, either through their use as probes capable of hybridizing to SSE complementary sequences or as primers for various amplification techniques, for example, polymerase chain reaction (PCR) cloning strategies.
  • PCR polymerase chain reaction
  • a combination of different ohgonucleotide probes may be used for the screening of a recombinant DNA library.
  • the oligonucleotides may be detectably-labeled using methods known in the art and used to probe filter replicas from a recombinant DNA library.
  • Recombinant DNA libraries are prepared according to methods well known in the art, for example, as described in Ausubel et al. (supra), or they may be obtained from commercial sources.
  • High stringency conditions may include hybridization at about 42 °C and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 1% SDS, 2X SSC, 10% Dextran sulfate, a first wash at about 65 °C, about 2X SSC, and 1% SDS, followed by a second wash at about 65 °C and about 0.1X SSC.
  • high stringency conditions may include hybridization at about 42 °C and about 50% formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5X SSPE, IX Denhardt's, followed by two washes at room temperature and 2X SSC, 0.1% SDS, and two washes at between 55- 60°C and 0.2X SSC, 0.1% SDS.
  • SSE genes having about 30% or greater sequence identity to the SSE genes described herein include, for example, hybridization at about 42 °C and 0.1 mg/mL sheared salmon sperm DNA, 1% SDS, 2X SSC, and 10% Dextran sulfate (in the absence of formamide), and a wash at about 37°C and 6X SSC, about 1% SDS.
  • the low stringency hybridization may be carried out at about 42 °C and 40%o formamide, 0.1 mg/mL sheared salmon sperm DNA, 0.5% SDS, 5X SSPE, IX Denhardt's, followed by two washes at room temperature and 2X SSC, 0.1% SDS and two washes at room temperature and 0.5X SSC, 0.1% SDS.
  • These stringency conditions are exemplary; other appropriate conditions may be determined by those skilled in the art.
  • RNA gel blot analysis of total or poly(A+) RNAs isolated from any plant may be used to determine the presence or absence of an SSE transcript using conventional methods.
  • SSE oligonucleotides may also be used as primers in amplification cloning strategies, for example, using PCR.
  • PCR methods are well known in the art and are described, for example, in PCR Technology, Erlich, ed., Stockton Press, London, 1989; PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc., New York, 1990; and Ausubel et al. (supra).
  • Primers are optionally designed to allow cloning ofthe amplified product into a suitable vector, for example, by including appropriate restriction sites at the 5' and 3' ends ofthe amplified fragment (as described herein).
  • SSE sequences may be isolated using the PCR "RACE” technique, or Rapid Amplification of cDNA Ends (see, e.g., Innis et al. (supra)).
  • ohgonucleotide primers based on an SSE sequence are oriented in the 3' and 5' directions and are used to generate overlapping PCR fragments. These overlapping 3'- and 5'-end RACE products are combined to produce an intact full-length cDNA. This method is described in Innis et al. (supra); and Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998, 1988.
  • any plant cDNA or cDNA expression library may be screened by functional complementation of an sse mutant (for example, the ssel mutant described herein) according to standard methods described herein.
  • Confirmation of a sequence's relatedness to the SSE polypeptide family may be accomplished by a variety of conventional methods including, but not limited to, functional complementation assays and sequence comparison ofthe gene and its expressed product.
  • the activity ofthe gene product may be evaluated according to any ofthe techniques described herein, for example, the functional or immunological properties of its encoded product.
  • SSE Polypeptide Expression SSE polypeptides may be expressed and produced by transformation of a suitable host cell with all or part of an SSE cDNA (for example, the SSE cDNA (SEQ ID NO:l) described above) in a suitable expression vehicle or with a plasmid construct engineered for increasing the expression of an SSE polypeptide (supra) in vivo.
  • SSE cDNA for example, the SSE cDNA (SEQ ID NO:l) described above
  • the SSE protein may be produced in a prokaryotic host, for example, E. coli, or in a eukaryotic host, for example, Saccharomyces cerevisiae, mammalian cells (for example, COS 1 or NIH 3T3 cells), or any of a number of plant cells or whole plant including, without limitation, algae, tree species, ornamental species, temperate fruit species, tropical fruit species, vegetable species, legume species, crucifer species, monocots, dicots, or in any plant of commercial or agricultural significance.
  • a prokaryotic host for example, E. coli
  • a eukaryotic host for example, Saccharomyces cerevisiae, mammalian cells (for example, COS 1 or NIH 3T3 cells), or any of a number of plant cells or whole plant including, without limitation, algae, tree species, ornamental species, temperate fruit species, tropical fruit species, vegetable species, legume species, crucifer species, monocots, dicots, or in any plant of
  • suitable plant hosts include, but are not limited to, conifers, petunia, tomato, potato, pepper, tobacco, Arabidopsis, grape, lettuce, sunflower, oilseed rape, flax, cotton, sugarbeet, celery, soybean, alfalfa, Medicago, lotus, Vigna, cucumber, carrot, eggplant, cauliflower, horseradish, morning glory, poplar, walnut, apple, grape, asparagus, cassava, rice, maize, millet, onion, barley, orchard grass, oat, rye, and wheat.
  • Such cells are available from a wide range of sources including the American Type Culture Collection (Rockland, MD); or from any of a number seed companies, for example, W. Atlee Burpee Seed Co. (Warminster, PA), Park Seed Co. (Greenwood, SC), Johnny Seed Co. (Albion, ME), or Northrup King Seeds (Harstville, SC).
  • Vasil I.K. Cell Culture and Somatic Cell Genetics of Plants, Vol I, II, III Laboratory Procedures and Their Applications Academic Press, New York, 1984; Dixon, R.A., Plant Cell Culture-A Practical Approach, IRL Press, Oxford University, 1985; Green et al., Plant Tissue and Cell Culture, Academic Press, New York, 1987; and Gasser and Fraley, Science 244:1293, 1989.
  • DNA encoding an SSE polypeptide is carried on a vector operably linked to control signals capable of effecting expression in the prokaryotic host.
  • the coding sequence may contain, at its 5' end, a sequence encoding any ofthe known signal sequences capable of effecting secretion ofthe expressed protein into the periplasmic space ofthe host cell, thereby facilitating recovery ofthe protein and subsequent purification.
  • Prokaryotes most frequently used are various strains of E. coli; however, other microbial strains may also be used.
  • Plasmid vectors are used which contain replication origins, selectable markers, and control sequences derived from a species compatible with the microbial host. Examples of such vectors are found in Pouwels et al.
  • prokaryotic control sequences are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences. Promoters commonly used to direct protein expression include the beta-lactamase (penicillinase), the lactose (lac) (Chang et al., Nature 198:1056, 1977), the tryptophan (Trp) (Goeddel et al, Nucl. Acids Res. 8:4057, 1980), and the tac promoter systems, as well as the lambda-derived P L promoter and N-gene ribosome binding site (Simatake et al., Nature 292:128, 1981).
  • SSE polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, WI). According to this expression system, DNA encoding an SSE polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the SSE gene is under the control ofthe T7 regulatory signals, expression of SSE is induced by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains which express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant SSE polypeptide is then isolated according to standard methods known in the art, for example, those described herein.
  • Another bacterial expression system for SSE polypeptide production is the pGEX expression system (Pharmacia).
  • This system employs a GST gene fusion system which is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products.
  • the protein of interest is fused to the carboxyl terminus ofthe glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione.
  • Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain.
  • proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.
  • the method of transformation or transfection and the choice of vehicle for expression ofthe SSE polypeptide will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; Gelvin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990; Kindle, K., Proc. Natl. Acad. Sci., U.S.A. 87:1228, 1990; Potrykus, I., Annu. Rev. Plant Physiol. Plant Mol.
  • Expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P.H. Pouwels et al., 1985, Supp. 1987); Gasser and Fraley (supra); Clontech Molecular Biology Catalog (Catalog 1992/93 Tools for the Molecular Biologist, Palo Alto, CA); and the references cited above. Other expression constructs are described by Fraley et al. (U.S. Pat. No. 5,352,605).
  • an SSE polypeptide is produced by a stably-transfected plant cell line, a transiently-transfected plant cell line, or by a transgenic plant.
  • a number of vectors suitable for stable or extrachromosomal transfection of plant cells or for the establishment of transgenic plants are available to the public; such vectors are described in Pouwels et al. (supra), Weissbach and Weissbach (supra), and Gelvin et al. (supra). Methods for constructing such cell lines are described in, e.g., Weissbach and Weissbach (supra), and Gelvin et al. (supra).
  • plant expression vectors include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker.
  • plant expression vectors may also contain, if desired, a promoter regulatory region (for example, one conferring inducible or constitutive, pathogen- or wound-induced, 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.
  • a promoter regulatory region for example, one conferring inducible or constitutive, pathogen- or wound-induced, environmentally- or developmentally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site for example, one conferring inducible or constitutive, pathogen- or wound-induced, environmentally- or developmentally-regulated, or cell- or tissue-specific expression
  • a transcription initiation start site for example, one conferring inducible or constitutive, pathogen-
  • the SSE DNA sequence ofthe invention may, if desired, be combined with other DNA sequences in a variety of ways.
  • the SSE DNA sequence of the invention may be employed with all or part ofthe gene sequences normally associated with the SSE protein.
  • a DNA sequence encoding an SSE protein is combined in a DNA construct having a transcription initiation control region capable of promoting transcription and translation in a host cell.
  • the constructs will involve regulatory regions functional in plants which provide for modified production of SSE protein as discussed herein.
  • the open reading frame coding for the SSE protein or functional fragment thereof will be joined at its 5' end to a transcription initiation regulatory region such as the sequence naturally found in the 5' upstream region ofthe SSE structural gene. Numerous other transcription initiation regions are available which provide for constitutive or inducible regulation.
  • 5' upstream non-coding regions are obtained from other genes, for example, from genes regulated during meristem development, seed development, embryo development, or leaf development.
  • Regulatory transcript termination regions may also be provided in DNA constructs of this invention as well.
  • Transcript termination regions may be provided by the DNA sequence encoding the SSE protein or any convenient transcription termination region derived from a different gene source.
  • the transcript termination region will contain preferably at least 1-3 kb of sequence 3' to the structural gene from which the termination region is derived.
  • Plant expression constructs having SSE as the DNA sequence of interest for expression may be employed with a wide variety of plant life, particularly plant life involved in the production of storage reserves (for example, those involving carbon and nitrogen metabolism).
  • plant life particularly plant life involved in the production of storage reserves (for example, those involving carbon and nitrogen metabolism).
  • Such genetically-engineered plants are useful for a variety of industrial and agricultural applications as discussed infra.
  • this invention is applicable to dicotyledons and monocotyledons, and will be readily applicable to any new or improved transformation or regeneration method.
  • the expression constructs include at least one promoter operably linked to at least one SSE gene.
  • An example of a useful plant promoter according to the invention is a caulimovirus promoter, for example, a cauliflower mosaic virus
  • CaMV CaMV promoter. These promoters confer high levels of expression in most plant tissues, and the activity of these promoters is not dependent on virally encoded proteins. CaMV is a source for both the 35S and 19S promoters. Examples of plant expression constructs using these promoters are found in Fraley et al., U.S. Pat. No. 5,352,605. In most tissues of transgenic plants, the CaMV 35S promoter is a strong promoter (see, e.g., Odell et al., Nature 313:810, 1985). The CaMV promoter is also highly active in monocots (see, e.g., Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen.
  • activity of this promoter can be further increased (i.e., between 2-10 fold) by duplication ofthe CaMV 35S promoter (see e.g., Kay et al, Science 236:1299, 1987; Ow et al., Proc. Natl. Acad. Sci., U.S.A. 84:4870, 1987; and Fang et al, Plant Cell 1 :141, 1989, and McPherson and Kay, U.S. Pat. No. 5,378,142).
  • Exemplary monocot promoters include, without limitation, commelina yellow mottle virus promoter, sugar cane badna virus promoter, rice tungro bacilliform virus promoter, maize streak virus element, and wheat dwarf virus promoter.
  • the SSE gene product in an appropriate tissue, at an appropriate level, or at an appropriate developmental time.
  • gene promoters each with its own distinct characteristics embodied in its regulatory sequences, shown to be regulated in response to inducible signals such as the environment, hormones, and/or developmental cues.
  • gene promoters that are responsible for heat-regulated gene expression (see, e.g., Callis et al., Plant Physiol. 88:965, 1988; Takahashi and Komeda, Mol. Gen. Genet. 219:365, 1989; and
  • hormone-regulated gene expression for example, the abscisic acid (ABA) responsive sequences from the Em gene of wheat described by Marcotte et al., Plant Cell 1 :969, 1989; the ABA- inducible HVA1 and HVA22, and rd29A promoters described for barley and
  • Plant expression vectors may also optionally include RNA processing signals, e.g, introns, which have been shown to be important for efficient RNA synthesis and accumulation (Callis et al., Genes and Dev. 1:1183, 1987).
  • introns RNA processing signals
  • Plant expression vectors may also optionally include RNA processing signals, e.g, introns, which have been shown to be important for efficient RNA synthesis and accumulation (Callis et al., Genes and Dev. 1:1183, 1987).
  • introns e.g, introns
  • an intron may be positioned upstream or downstream of an SSE polypeptide-encoding sequence in the transgene to modulate levels of gene expression.
  • the expression vectors may also include regulatory control regions which are generally present in the 3' regions of plant genes (Thornburg et al., Proc. Natl. Acad. Sci. U.S.A. 84:744, 1987; An et al., Plant Cell 1 :115, 1989).
  • the 3' terminator region may be included in the expression vector to increase stability ofthe mRNA.
  • One such terminator region may be derived from the PI-II terminator region of potato.
  • other commonly used terminators are derived from the octopine or nopaline synthase signals.
  • the plant expression vector also typically contains a dominant selectable marker gene used to identify those cells that have become transformed.
  • Useful selectable genes for plant systems include genes encoding antibiotic resistance genes, for example, those encoding resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin. Genes required for photosynthesis may also be used as selectable markers in photosynthetic-deficient strains. Finally, genes encoding herbicide resistance may be used as selectable markers; useful herbicide resistance genes include the bar gene encoding the enzyme phosphinothricin acetyltransferase and conferring resistance to the broad spectrum herbicide Basta® (Hoechst AG, Frankfurt, Germany).
  • Efficient use of selectable markers is facilitated by a determination ofthe susceptibility of a plant cell to a particular selectable agent and a determination of the concentration of this agent which effectively kills most, if not all, ofthe transformed cells.
  • Some useful concentrations of antibiotics for tobacco transformation include, e.g., 75-100 ⁇ g/mL (kanamycin), 20-50 ⁇ g/mL (hygromycin), or 5-10 ⁇ g/mL
  • the plant expression construct may contain a modified or fully-synthetic structural SSE coding sequence which has been changed to enhance the performance ofthe gene in plants.
  • Methods for constructing such a modified or synthetic gene are described in Fischoff and Perlak, U.S. Pat. No. 5,500,365.
  • gene targeting can be used to silence or replace the endogenous gene with an engineered allele; thus the phenotype of the altered gene, or its regulatory sequences, can be evaluated inplanta.
  • methods for constructing transgene constructs for silencing or inactivating gene expression in plants using antisense or co-suppression technologies are well known in the art. It should be readily apparent to one skilled in the art of molecular biology, especially in the field of plant molecular biology, that the level of gene expression of a transgene construct is dependent, not only on the combination of promoters, RNA processing signals, and terminator elements, but also on how these elements are used to increase the levels of selectable marker gene expression.
  • Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes) (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol 6, PWJ Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P., and Draper, J,. In: DNA Cloning, Vol II, D.M.
  • Suitable plants for use in the practice ofthe invention include, but are not limited to, sugar cane, wheat, rice, maize, sugar beet, potato, barley, manioc, sweet potato, soybean, sorghum, cassava, banana, grape, oats, tomato, millet, coconut, orange, rye, cabbage, apple, watermelon, canola, cotton, carrot, garlic, onion, pepper, strawberry, yam, peanut, onion, bean, pea, mango, citrus plants, walnuts, and sunflower.
  • the following is an example outlining one particular technique, an Agrobacterium-mediated plant transformation.
  • the general process for manipulating genes to be transferred into the genome of plant cells is carried out in two phases. First, cloning and DNA modification steps are carried out in E. coli, and the plasmid containing the gene construct of interest is transfe ⁇ ed by conjugation or electroporation into Agrobacterium. Second, the resulting Agrobacterium strain is used to transform plant cells.
  • the plasmid contains an origin of replication that allows it to replicate in Agrobacterium and a high copy number origin of replication functional in E. coli. This permits facile production and testing of transgenes in E.
  • Resistance genes can be carried on the vector, one for selection in bacteria, for example, streptomycin, and another that will function in plants, for example, a gene encoding kanamycin resistance or herbicide resistance.
  • restriction endonuclease sites for the addition of one or more transgenes and directional T-DNA border sequences which, when recognized by the transfer functions of Agrobacterium, delimit the DNA region that will be transfe ⁇ ed to the plant.
  • plant cells may be transformed by shooting into the cell tungsten microprojectiles on which cloned DNA is precipitated.
  • a gunpowder charge 22 caliber Power Piston Tool Charge
  • an air-driven blast drives a plastic macroprojectile through a gun ba ⁇ el.
  • An aliquot of a suspension of tungsten particles on which DNA has been precipitated is placed on the front ofthe plastic macroprojectile.
  • the latter is fired at an acrylic stopping plate that has a hole through it that is too small for the macroprojectile to pass through.
  • the plastic macroprojectile smashes against the stopping plate, and the tungsten microprojectiles continue toward their target through the hole in the plate.
  • the target can be any plant cell, tissue, seed, or embryo.
  • Plant cells transformed with a plant expression vector can be regenerated, for example, from single cells, callus tissue, or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant; such techniques are described, e.g., in Vasil supra; Green et al., supra; Weissbach and Weissbach, supra; and Gelvin et al., supra.
  • a cloned SSE polypeptide construct under the control ofthe 35S CaMV promoter and the nopaline synthase terminator and carrying a selectable marker (for example, kanamycin resistance) is transformed into Agrobacterium. Transformation of leaf discs (for example, of tobacco or potato leaf discs), with vector-containing Agrobacterium is carried out as described by Horsch et al. (Science 227:1229, 1985). Putative transformants are selected after a few weeks (for example, 3 to 5 weeks) on plant tissue culture media containing kanamycin (e.g. 100 ⁇ g/mL). Kanamycin-resistant shoots are then placed on plant tissue culture media without hormones for root initiation.
  • kanamycin resistance for example, kanamycin resistance
  • Kanamycin-resistant plants are then selected for greenhouse growth. If desired, seeds from self- fertilized transgenic plants can then be sowed in a soil-less medium and grown in a greenhouse. Kanamycin- resistant progeny are selected by sowing surfaced sterilized seeds on hormone-free kanamycin-containing media. Analysis for the integration ofthe transgene is accomplished by standard techniques (see, for example, Ausubel et al. supra; Gelvin et al. supra).
  • Transgenic plants expressing the selectable marker are then screened for transmission ofthe transgene DNA by standard immunoblot and DNA detection techniques. Each positive transgenic plant and its transgenic progeny are unique in comparison to other transgenic plants established with the same transgene.
  • transgene DNA Integration ofthe transgene DNA into the plant genomic DNA is in most cases random, and the site of integration can profoundly affect the levels and the tissue and developmental patterns of transgene expression. Consequently, a number of transgenic lines are usually screened for each transgene to identify and select plants with the most appropriate expression profiles. Transgenic lines are evaluated for levels of transgene expression.
  • RNA expression at the RNA level is determined initially to identify and quantitate expression-positive plants.
  • Standard techniques for RNA analysis are employed and include PCR amplification assays using ohgonucleotide primers designed to amplify only transgene RNA templates and solution hybridization assays using transgene- specific probes (see, e.g., Ausubel et al., supra).
  • the RNA-positive plants are then analyzed for protein expression by Western immunoblot analysis using SSE specific antibodies (see, e.g., Ausubel et al., supra).
  • in situ hybridization and immunocytochemistry can be done using transgene- specific nucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue.
  • the recombinant SSE protein may be expressed in any cell or in a transgenic plant (for example, as described above), it may be isolated, e.g., using affinity chromatography.
  • an anti-SSE polypeptide antibody e.g., produced as described in Ausubel et al., supra, or by any standard technique
  • Lysis and fractionation of SSE-producing cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra).
  • the recombinant protein can, if desired, be further purified, for example, by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
  • Polypeptides ofthe invention can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, IL). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful SSE fragments or analogs.
  • Engineering Storage Reserve Materials e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, IL.
  • SSE genes that are isolated from a host plant may be engineered for increased or decreased expression in the same plant, a closely related species, or a distantly related plant species.
  • a host plant e.g., Arabidopsis or Brassica
  • the cruciferous Arabidopsis SSEl gene may be engineered for constitutive expression and then transformed into an Arabidopsis host plant.
  • the Arabidopsis SSEl gene may be engineered for expression other cruciferous plants, such as the Brassicas (for example, broccoli, cabbage, and cauliflower). Evaluation ofthe modification confe ⁇ ed on a plant by ectopic expression of an SSE gene is determined according to conventional methods and assays (for example, those described herein).
  • constitutive expression ofthe SSEl gene of Arabidopsis (Fig. 2A; SEQ ID NO:l) is used to alter seed storage reserve deposition in transgenic seeds of Brassica.
  • a plant expression vector is constructed that contains an SSEl cDNA sequence expressed under the control of the enhanced CaMV 35S promoter as described by McPherson and Kay (U.S. Patent No. 5,359,142). This expression vector is then used to transform Brassica according to the methods described in Moloney et al. (U.S. Patent No. 5,750,827). Seeds of transformed Brassica and control plants are then profiled for storage reserve material according to conventional methods to determine the qualitative and quantitative aspects ofthe deposited reserve materials. Transformed plants that express an SSEl gene and produce seeds having an increased level of storage reserve material (e.g., reserve lipid or storage protein) relative to control plants are taken as being useful in the invention.
  • storage reserve material e.g., reserve lipid or storage protein
  • gene silencing or inactivation technologies may also be used to modify or alter the deposition of seed storage reserve material.
  • Exemplary methods for silencing or inactivating plant gene expression include antisense RNA (Shewmaker et al., U.S. Patent 5,107,065), co-suppression (Napoli et al, U.S. Patent 5,034,327), and homologous recombination (Offringa et al., United States Patent 5,501,967).
  • a plant expression vector is constructed that contains an antisense SSEl which is expressed under the control ofthe enhanced CaMV 35S promoter as described by McPherson and Kay, supra and Shewmaker at al. (U.S. Patent No. 5,107,065).
  • This expression vector is then used to transform Brassica according to the methods described in Moloney et al., supra. To assess reserve material deposition, transformed plants and appropriate controls are grown, and the storage reserves of their seeds are evaluated according to standard methods, for example, those described herein. Transformed Brassica plants that express an antisense SSEl sequence and that produce seeds having a decreased level of reserve material relative to control plants are taken as being useful in the invention. Engineering Stress-Protected Plants
  • constructs designed for the expression of an SSE polypeptide are useful for generating transgenic seeds having an increased level of tolerance to environmental stress. To achieve such tolerance, it is important to express such a protein at an effective level in a transgenic seed. Seed-specific gene promoters are especially useful for this purpose. Evaluation ofthe level of stress protection confe ⁇ ed to a seed by expression of a DNA sequence expressing an SSEl polypeptide is determined according to conventional methods and assays as described below.
  • seed-specific expression of an SSE gene for example, the SSEl
  • Brassica to enhance salt stress tolerance.
  • a plant expression vector is constructed that contains an SSEl sequence expressed under the control of a Brassica seed-specific promoter. This expression vector is then used to transform Brassica according to standard methods.
  • seeds obtained from transformed Brassica plants and appropriate controls are evaluated according to standard methods.
  • Transgenic seeds containing the gene are germinated in the presence of various salt or osmotically active solutions to determine whether transgenic seeds demonstrate increased tolerance or resistance to salt stress.
  • seedlings can also be grown in hydroponic systems and challenged with salt or agents of differing osmotic potentials at different, or all, developmental stages in order to assess the response of SSEl -expressing plants to these stresses. Growth and physiological measurements are used to document the differences. Transformed Brassica plants which produce seeds having an increased level of salt tolerance relative to control plants are taken as being useful in the invention. Engineering Plants Having Increased Yield/Productivity
  • Seeds of transgenic plants expressing a recombinant SSE gene are planted out in test plots, and their agronomic performance is compared to standard plants using techniques familiar to those of skill in the art. Optionally included in this comparison are plants of similar genetic background without the transgene. A yield benefit is observed and plants exhibiting the increased yield are advanced for commercialization.
  • transgenic plants expressing an SSE gene are field tested for agronomic performance under conditions, including, but not limited to, limited or inadequate water availability.
  • transgenic plants expressing the SSEl gene exhibit higher yield than their non-transgenic counterparts under non-optimal growing conditions.
  • SSE sequences also facilitates the identification of polypeptides which interact with the SSE protein.
  • polypeptide-encoding sequences are isolated by any standard two hybrid system (see, for example, Fields et al., Nature 340:245-246, 1989; Yang et al., Science 257:680-682, 1992; Zervos et al., Cell 72:223-232, 1993).
  • all or a part ofthe SSE sequence may be fused to a DNA binding domain (such as the GAL4 or LexA DNA binding domain).
  • a reporter gene for example, a lacZ or LEU2 reporter gene bearing appropriate DNA binding sites, this fusion protein is used as an interaction target.
  • Candidate interacting proteins fused to an activation domain are then co-expressed with the SSE fusion in host cells, and interacting proteins are identified by their ability to contact the SSE sequence and stimulate reporter gene expression. SSE-interacting proteins identified using this screening method provide good candidates for proteins that are involved in the acquired resistance signal transduction pathway.
  • SSE polypeptides described herein may be used to raise antibodies useful in the invention; such polypeptides may be produced by recombinant or peptide synthetic techniques (see, e.g., Solid Phase Peptide Synthesis, 2nd ed., 1984, Pierce Chemical Co., Rockford, IL; Ausubel et al., supra).
  • the peptides may be coupled to a carrier protein, such as KLH as described in Ausubel et al, supra.
  • the KLH-peptide is mixed with Freund's adjuvant and injected into guinea pigs, rats, or preferably rabbits.
  • Antibodies may be purified by peptide antigen affinity chromatography.
  • Monoclonal antibodies may be prepared using the SSE polypeptides described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981; Ausubel et al., supra).
  • polyclonal or monoclonal antibodies are tested for specific SSE recognition by Western blot or immunoprecipitation analysis (by the methods described in Ausubel et al., supra).
  • Antibodies which specifically recognize SSE polypeptides are considered to be useful in the invention; such antibodies may be used, e.g., in an immunoassay to monitor the level of SSE polypeptide produced by a plant.
  • the invention further includes analogs of any naturally-occurring plant SSE polypeptide.
  • Analogs can differ from the naturally-occurring SSE protein by amino acid sequence differences, by post-translational modifications, or by both.
  • Analogs ofthe invention will generally exhibit at least 40%>, more preferably 50%), and most preferably 60%> or even having 70%>, 80%>, or 90% identity with all or part of a naturally-occurring plant SSE amino acid sequence.
  • the length of sequence comparison is at least 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues.
  • Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • Analogs can also differ from the naturally-occurring SSE polypeptide by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethyl methylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, supra, or Ausubel et al, supra).
  • cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., ⁇ or ⁇ amino acids.
  • the invention also includes SSE polypeptide fragments.
  • fragment means at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least 60 to 80 or more contiguous amino acids. Fragments of SSE polypeptides can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
  • an SSE polypeptide fragment includes an ankyrin-repeat motif as described herein.
  • an SSE fragment is capable of interacting with a second polypeptide component ofthe SSE signal transduction cascade.
  • the invention includes nucleotide sequences that facilitate specific detection of an SSE nucleic acid.
  • SSE sequences described herein or portions thereof may be used as probes to hybridize to nucleotide sequences from other plants (e.g., dicots, monocots, gymnosperms, and algae) by standard hybridization techniques under conventional conditions. Sequences that hybridize to an SSE coding sequence or its complement and that encode an SSE polypeptide are considered useful in the mvention.
  • fragment means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and most preferably at least 40 to 80 or more contiguous nucleotides. Fragments of SSE nucleic acid sequences can be generated by methods known to those skilled in the art. All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
  • An isolated nucleic acid molecule comprising a sequence encoding an SSE polypeptide.
  • nucleic acid molecule of claim 1 wherein said sequence encodes an SSE polypeptide having at least 30%> identity with the amino acid sequence shown in Fig. 2B (SEQ ID NO:2).
  • nucleic acid molecule of claim 1 wherein said sequence encodes an SSE polypeptide that, when expressed in a cell of a plant, modifies the production of food storage reserves.
  • nucleic acid molecule of claim 1 wherein said sequence encodes an SSE polypeptide that, when expressed in a cell of a plant, facilitates the intracellular transport of a storage protein.
  • nucleic acid molecule of claim 1 wherein said sequence encodes an SSE polypeptide that, when expressed in a cell of a plant, facilitates the formation of protein bodies.
  • nucleic acid molecule of claim 1 wherein said sequence encodes an SSE polypeptide that, when expressed in a cell of a plant, facilitates the formation of oil bodies.
  • nucleic acid molecule of claim 1 wherein said nucleic acid molecule is cDNA.

Abstract

Cette invention concerne un gène responsable de la biogenèse des protéines et des lipides. L'invention concerne également des méthodes qui permettent de produire des plantes présentant un ou plusieurs traits phénotypiques requis en rapport avec un matériau de réserve.
PCT/US2000/009192 1999-04-08 2000-04-07 Modification des reserves des plantes WO2000061735A1 (fr)

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