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WO1999023200A2 - Gene de deshydrogenase de glucose de disphosphate d'uridine, et role que joue ce gene dans la modification de la croissance et des caracteristiques des plantes - Google Patents

Gene de deshydrogenase de glucose de disphosphate d'uridine, et role que joue ce gene dans la modification de la croissance et des caracteristiques des plantes Download PDF

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WO1999023200A2
WO1999023200A2 PCT/US1998/022964 US9822964W WO9923200A2 WO 1999023200 A2 WO1999023200 A2 WO 1999023200A2 US 9822964 W US9822964 W US 9822964W WO 9923200 A2 WO9923200 A2 WO 9923200A2
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plant
udp
nucleic acid
cell
dehydrogenase
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PCT/US1998/022964
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English (en)
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Grant Cramer
Raimund Tenhaken
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The Board Of Regents Of The University And Community College System Of Nevada On Behalf Of The University Of Nevada-Reno
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Priority to AU13681/99A priority Critical patent/AU1368199A/en
Publication of WO1999023200A2 publication Critical patent/WO1999023200A2/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N15/8261—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

  • the present invention pertains, in general, to nucleic acids encoding plant derived uridine diphosphate glucose (UDP-Glu) dehydrogenase.
  • the present invention pertains to modifying the genotypes of plant cells to alter the cell wall composition of plant cells transformed with nucleic acids encoding UDP-Glu dehydrogenase.
  • the plant cell cannot expand for very long without a supply of building materials for the cell wall.
  • Synthases are intimately connected with the endomembrane system and probably form a complex of enzymes performing sequential steps in the synthesis of polysaccharide. These are important enzymes but are presently impossible to assay since there are no good in vitro assays to date which are good indicators of in vivo activity.
  • Cellulose and callose are the only polysaccharides known to be made at the plasma membrane (Delmer and Amor, 1995), whereas the interlocking glycans and pectins are synthesized, and modified in the Golgi apparatus, packaged in secretory vesicles and transported to the cell wall (Driouch, Faye and Staehelin, 1993; Gibeaut and Carptia, 1994).
  • Cell expansion is primarily controlled by wall-loosening events, which may be the result of enzymatic action in the cell wall or the secretion of materials to build the cell wall.
  • wall-loosening events which may be the result of enzymatic action in the cell wall or the secretion of materials to build the cell wall.
  • Ray (1987) discussed the importance of biosynthetic incorporation of wall matrix polymers and their role in the expansion of plant cell walls. He showed how a supply of substrates may directly affect cell wall loosening, particularly since it is the inner cell wall layers which are mechanically the most important layers in regard to cell wall expansion. A greater supply of substrates leads to increased cell wall loosening and increased cell expansion.
  • Polysaccharides represent 90% of the cell wall on a dry weight basis. The remaining 5 - 10 % of the wall is protein.
  • Cellulose accounts for 25-30% of the wall and is synthesized at the plasma membrane.
  • Matrix polysaccharides composed of hemicelluloses and pectins, account for 15-25% and 35% of the wall, respectively, and are synthesized in the Golgi apparatus.
  • biosynthesis and secretion of molecules to the cell wall is related to cell expansion (Hetherington and Fry, 1993; Kutschera and Briggs, 1987; Ray, 1987; Schindler et al, 1994).
  • Auxin a plant growth hormone, stimulates cell expansion, hemicellulose incorporation into the cell wall and cell wall extensibility (Kutschera and Briggs, 1987); whereas, abscisic acid (ABA), a plant-stress hormone, and salinity inhibit hemicellulose incorporation and cell expansion (Wakabayashi, Sakurai and Kuraishi, 1991; Zhong and Lauchli, 1988; Zhong and Lauchli, 1993). Furthermore, treatment with monensin (Kutschera and Briggs,
  • Brefeldin-A increases the yield threshold of the cell wall (a hardening process) in a similar manner as salinity and ABA. When the cell wall hardens, it makes it more difficult for the wall to expand, consequently slowing down growth.
  • UDP-Glc is the precursor molecule for synthesis of the majority of glycosyl units of structural carbohydrates ( Figure 1).
  • Uridine Diphosphate Glucose Dehydrogenase also known as UDP-Glc dehydrogenase or UDP-Glc DH, is the precursor molecule for synthesis of the majority of glycosyl units of structural carbohydrates (Gibeaut and Carpita, 1994).
  • the interconversion of nucleotide sugars begins with the activity of UDP-Glc dehydrogenase, which oxidizes UDP-Glc to UDP-GlcUA.
  • UDP-GlcUA can then be converted to UDP-Xyl through the activity of UDP-GlcUA decarboxylase.
  • a set of reversible epimerases forms UDP-Gal, UDP- GalUA and UDP-Ara. Because UDP-Glc dehydrogenase operates far from equilibrium and because so much of the cell wall carbohydrate is acted upon by this enzyme, it is has been speculated to be a good candidate for a control point in the metabolic pathway of cell wall synthesis (Amino et al., 1985; Dalessandro and Northcote, 1997; Robertson and McCormack, 1995).
  • the present invention involves the isolation, characterization and utilization of the cDNA product for UDP-Glc dehydrogenase in soybeans and the process of altering plant growth by modulating the levels and/or activity of this enzyme in plants.
  • the cDNA of UDP-Glc dehydrogenase may be used to identify other similar genes in other plant species.
  • the insertion of sense and antisense cDNA of UDP-Glc dehydrogenase into plants produces seeds and plants for numerous commercial purposes. An increase in the biosynthesis of cell walls, by increasing the activity of UDP-Glc dehydrogenase, will lead to an increase in plant growth and crop yields, especially under conditions of increased CO 2 levels or increased salinity. Other benefits will also be obtained by inserting additional copies of the gene for UDP-Glc dehydrogenase into plants.
  • UDP-Glc dehydrogenase alters the composition of the cell wall, yielding an increase in the proportion of non-cellulosic polysaccharides. This in turn will alter the properties of the cell wall and will have widespread applications.
  • the processes of the present invention enable one skilled in the art to alter the texture of foods and provides an improved animal feed by reducing the levels of phenolics in the cell wall, which interfere with animal digestion.
  • the tensile strength and flexibility of textiles and industrial materials, such as lumber and other plant products, may also be altered using the processes of the present invention.
  • the ability of the plant to defend itself against plant pathogens and insects may be altered using the methods disclosed herein.
  • a reduction in the activity of UDP-Glc dehydrogenase by the insertion of antisense DNA of UDP-Glc dehydrogenase should also alter the properties of the cell wall, thus affecting food texture, plant defense and industrial materials, as described herein.
  • a reduction in UDP-Glc dehydrogenase activity may provide a new process for developing dwarf plants for use in the agricultural and horticultural industries.
  • This invention comprises methods of altering cell wall composition by expression of exogenous nucleic acids encoding UDP-Glu dehydrogenase or functional fragments thereof in plants and plant cells.
  • this invention provides a method of introducing into a plant cell nucleic acids having a sequence encoding a plant derived UDP-Glu dehydrogenase.
  • Figure 1 Pathway for polysaccharide biosynthesis: a) synthesis of UDP-sugars; b) interconversion of UDP-sugars, and c) synthesis of polysaccharides.
  • Figure 2 Nucleotide sequence of the UDP-GlcDH from soybean (SEQ ID NO:l).
  • FIG. 3 Expression of UDP-GlcDH in soybean seedlings and plants.
  • Total RNA (10 ⁇ g) from different developmental stages of the plant was separated on a denaturing agarose gel, transferred to a nylon membrane, and hybridized with a 32 P-labeled cDNA probe for UDP- GlcDH.
  • RNA was prepared from plant organs as indicated in the figure.
  • C to E represents a 2-day-old seedling; D represents a 7-day-old plants; E shows a 2-week-old plant.
  • FIG. 1 Western blot analysis of wildtype tobacco and 4 independently-transformed lines overexpressing soybean UDP-Glc dehydrogenase.
  • Figure 5 Typical growth response of tobacco leaves of plants overexpressing UDP-Glc dehydrogenase as indicated with this example using leaf 3. Plants were grown hydroponically in growth chambers set to 16-h day/night cycles (300 ⁇ mol m '2 s '1 PAR) and 28/23 °C day/night temperatures.
  • Figure 6 Leaf area gain of wild type and line 2. Plants were grown hydroponically in growth chambers set to 16-h day/night cycles (300 ⁇ mol m 2 s "1 PAR) and 28/23 °C day/night temperatures.
  • A) Semi-log plot of changes in total leaf area with time. Data are means ⁇ SE; n 7 for wildtype and line 2. Symbols without error bars indicate that the SE was smaller than the size of the symbol.
  • Figure 7 Dry weight gain of wildtype and line 2 over time. Plants were grown hydroponically in growth chambers set to 16-h day/night cycles (300 ⁇ mol m 2 s '1 PAR) and
  • A) Semi-log plot of changes in total plant dry weight with time. Data are means ⁇ SE; n 7 for wildtype and line 2. Symbols without error bars indicate that the SE was smaller than the size of the symbol.
  • Figure 8 Comparison of average sized, 20-day-old tobacco plants. Wild type and line 2, a transgenic line overexpressing UDP-Glc dehydrogenase.
  • FIG. 9 Alignment of UDP-Glc dehydrogenase from soybean, Arabidopsis, and bovine liver. Identical sequences are white in black boxes (Tenhaken et al., Plant Physiol (1996) 112: 1127-1134, fully incorporated by reference).
  • FIG. 10 Genomic Southern blot with soybean and Arabidopsis DNA, restricted with EcoRI ( ⁇ ) or Xbal (X). The membrane was hybridized with the 32 P-labeled soybean cDNA probe.
  • A Soybean DNA (high stringency, 65 °C);
  • B soybean DNA (lower stringency, 58°C);
  • C Arabidopsis DNA (lower stringency, 58 °C). The sizes of the fragments are indicated in kilobase pairs (kBp).
  • UDP-Glc dehydrogenase As discussed above, a cDNA encoding soybean (Glycine max [L.] Merr.) UDP-Glc dehydrogenase has been cloned recently, and UDP-Glc dehydrogenase mRNA was shown to be highly expressed in the growing portions of leaves and roots of soybean (Tenhaken and Thulke, 1996). The present invention is particularly directed to such cDNAs and to their use to modulate the growth of plants, for example, soybean.
  • plant genes coding for UDP-Glc dehydrogenase include the specifically identified and characterized variants herein described as well as allelic variants, conservative substitution variants and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined below.
  • conservative variation or “conservative substitution” denote the replacement of an amino acid residue by another, biologically similar residue. Conservative variations or substitutions are not likely to change the shape of the polypeptide chain. Examples of conservative variations, or substitutions, include the replacement of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine, and the like. Therefore, all conservative substitutions are included in the invention as long as the plant UDP-Glc dehydrogenase polypeptide encoded by the nucleotide sequence is functionally unchanged or similar.
  • expression refers to the transcription and translation of a structural gene so that a protein is synthesized.
  • operably linked refers to functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates transcription of RNA corresponding to the second sequence.
  • promoter refers to a region of DNA upstream from the structural gene and involved in recognition and binding RNA polymerase and other proteins to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • mutation variously refers to the process by which a gene undergoes a structural change, a modified gene resulting from such a change or, by extension, an individual manifesting the mutation.
  • Mutation breeding is a process well known to one of ordinary skill in the plant breeding art whereby mutations are induced by mutagens in an effort to develop new crop varieties that can increase agricultural productivity.
  • a mutagen is any physical (e.g., ionizing radiation) or chemical agent (e.g., ethyl methanesulfonate) that raises the frequency of mutation above the spontaneous rate of natural mutations.
  • nucleic acid is defined as RNA or DNA (including cDNA and genomic DNA) which encode plant UDP-Glc dehydrogenase peptides, or which are complimentary to nucleic acids encoding such peptides, or which hybridize to such nucleic acids and remain stably bound to them under stringent conditions.
  • nucleic acids encode polypeptides sharing at least 60% sequence identity, more preferably at least 70% sequence identity, and even more preferably at least 80% sequence identity with plant UDP-Glc dehydrogenase peptide sequences.
  • plant or “plant-derived” UDP-Glc dehydrogenase genes or (UDP-GlcDH genes) include all naturally occurring homologs of the soybean UDP-GlcDH gene specifically disclosed herein.
  • plant UDP-Glc dehydrogenase proteins includes all naturally occurring allelic variants of a plant UDP-Glc dehydrogenase proteins that possess normal plant UDP-Glc dehydrogenase activity as demonstrated herein. In general, allelic variants of plant UDP-Glc dehydrogenase proteins will have a slightly different amino acid sequence than that specifically encoded by the UDP-Glc dehydrogenase proteins of the soybean UDP-Glc dehydrogenase gene.
  • the terms "soybean” or “soybean-derived" UDP-Glc dehydrogenase genes (or UDP- GlcDH genes) include all naturally occurring allelic variants of the soybean (Glycine max) UDP-
  • allelic variants of the soybean UDP-Glc dehydrogenase genes will have a slightly different amino acid sequence than that specifically encoded by the soybean UDP-Glc dehydrogenase genes of soybean.
  • soybean plant UDP-Glc dehydrogenase proteins includes all naturally occurring allelic variants of the soybean plant UDP-Glc dehydrogenase protein that possess normal plant
  • allelic variants of the plant UDP-Glc dehydrogenase protein will have a slightly different amino acid sequence than that specifically encoded by the nucleic acid sequence of SEQ ID NO: 1 but will be able to produce a protein possessing substantially equivalent activities.
  • an allelic variant could possess a slightly different amino acid sequence than those recited above, but could possess the ability to produce transformed plants which exhibit different levels of UDP-Glc dehydrogenase activity.
  • allelic variants of the plant UDP-Glc dehydrogenase protein will contain conservative amino acid substitutions from the plant UDP-Glc dehydrogenase sequences herein described or will contain a substitution of an amino acid from a corresponding position in a plant UDP-Glc dehydrogenase protein homolog (a plant UDP-Glc dehydrogenase protein isolated from an organism other than soybean, such as maize or tobacco).
  • stringent conditions are conditions in which hybridization yields a clear and readable hybridization signal. Stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl, 0.0015 M sodium citrate, 0.1 % SDS buffer at 50 °C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 40 °C.
  • formamide for example, 50% formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 40 °C.
  • Another example is using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS and 10% dextran sulfate at 40°C, with washes at 40°C in 0.2x SSC and 0.1% SDS.
  • 5x SSC 0.75 M NaCl, 0.075 M sodium citrate
  • 50 mM sodium phosphate pH 6.8
  • 0.1 % sodium pyrophosphate 5x Denhardt's solution
  • sonicated salmon sperm DNA 50 ⁇ g/ml
  • 0.1% SDS 0.1% dextran sulfate at 40°C
  • substantially pure is used herein to describe plant UDP-Glc dehydrogenase in that has been separated from the native contaminants or components that naturally accompany it.
  • a monomeric protein is substantially pure when at least about 60 to 70%) of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications typically share approximately the same polypeptide sequence.
  • a substantially pure protein will typically comprise over about 85 to 90% of a protein sample, preferably will comprise at least about 95%, and more preferably will be over about 99% pure. Purity is typically measured on a polyacrylamide gel, with homogeneity determined by staining. For certain purposes, high resolution will be desired and HPLC or a similar means for purification utilized.
  • a simple chromatography column or polyacrylamide gel will be used to determine purity. Whether soluble or membrane bound, the present invention provides for substantially pure preparations.
  • Various methods for their isolation from biological material may be devised, based in part upon the structural and functional descriptions contained herein.
  • a protein that is chemically synthesized or synthesized in a cellular system that is different from the cell from which it naturally originates will be substantially pure.
  • the term is also used to describe proteins and nucleic acids that have been synthesized in heterologous mammalian cells, bacterial cells such as E. coli and other prokaryotes.
  • nucleic acid will be nucleic acids that are identified and separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid.
  • the nucleic acid may be labeled for diagnostic and probe purposes, using any label known and described in the art as useful in connection with diagnostic assays. Accordingly, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a soybean UDP-Glc dehydrogenase gene or a fragment thereof.
  • the invention also includes degenerate sequences of the DNA as well as sequences that are substantially homologous.
  • the exemplified source of the UDP-Glc dehydrogenase for the invention is soybean, although UDP-Glc dehydrogenase genes from any plant source is encompassed by the invention.
  • the invention encompasses the nucleic acids for the UDP-
  • Glc dehydrogenase of any other monocotyledonous or dicotyledonous plant species including soybean, maize, Arabidopsis and tobacco.
  • the UDP-Glc dehydrogenase gene is most likely ubiquitous in the plant kingdom.
  • the nucleic acid molecule or fragment thereof may also be synthesized using methods known in the art. It is also possible to produce the molecule by genetic engineering techniques, by constructing DNA using any accepted technique, cloning the DNA in an expression vehicle and transfecting the vehicle into a cell which will express the compound.
  • polynucleotides encoding all or a portion of plant UDP-Glc dehydrogenase are also contemplated herein, as long as they encode a polypeptide with the functional activities of plant UDP-Glc dehydrogenase.
  • Polynucleotide sequences of the invention include DNA, cDNA, synthetic DNA and RNA sequences which encode plant UDP- Glc dehydrogenase. Such polynucleotides also include naturally occurring, synthetic and intentionally manipulated polynucleotides.
  • such polynucleotide sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly A sequences. As another example, portions of the mRNA sequence may be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription. As yet another example, plant UDP-Glc dehydrogenase polynucleotides may be subjected to site-directed mutagenesis.
  • the polynucleotides of the invention further include sequences that are degenerate as a result of the genetic code.
  • the genetic code is said to be degenerate because more than one nucleotide triplet codes for the same amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences, some bearing minimal nucleotide sequence homology to the nucleotide sequence of SEQ ID NO:l may be produced as a result of this invention.
  • nucleotide sequences are included in the invention as long as the amino acid sequence of plant UDP-Glc dehydrogenase polypeptide encoded by the nucleotide sequence is functionally unchanged or substantially similar in function.
  • the invention specifically contemplated each and every possible variation of peptide or nucleotide sequence that could be made by selecting combinations based on the possible amino acid and codon choices made in accordance with the standard triplet genetic code as applied to the plant UDP-Glc dehydrogenase sequences of the invention, as exemplified by SEQ ID NO:l, and all such variations are to be considered specifically disclosed herein.
  • fragments portions, segments of the sequences disclosed herein which selectively hybridize to the sequence of plant UDP-Glc dehydrogenase.
  • Selective hybridization refers to hybridization under stringent conditions (See, for example, the techniques in Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 1989), which distinguishes related from unrelated nucleotide sequences.
  • the active fragments of the invention which are complementary to mRNA and the coding strand of DNA, are usually at least about 15 nucleotides, more usually at least 20 nucleotides, preferably 30 nucleotides and more preferably may be 50 nucleotides or more.
  • the present invention provides nucleic acid molecules encoding plant UDP-Glc dehydrogenase proteins which hybridize with nucleic acid molecules comprising sequences complimentary to soybean UDP-Glc dehydrogenase under conditions of sufficient stringency to produce a clear signal.
  • Plants naturally contain a wildtype plant UDP-Glc dehydrogenase gene that codes for wildtype plant UDP-Glc dehydrogenase protein.
  • Wildtype when referring to nucleic acid sequences or protein sequences, means the genetic constitution of an organism in which a number of mutations (markers) may already exist at the start of a program of mutagenesis before further changes are introduced.
  • the wildtype plant UDP-Glc dehydrogenase protein refers to the various forms of plant UDP-Glc dehydrogenase protein found naturally before the introduction of a nucleotide sequence coding for the plant UDP-Glc dehydrogenase of the wildtype plant UDP-Glc dehydrogenase gene.
  • polypeptides of the present invention include the protein encoded by the plant UDP- Glc dehydrogenase nucleic acid sequence of SEQ ID NO:l, as well as polypeptides and fragments, particularly those which have the biological activity of plant UDP-Glc dehydrogenase and also those which have at least 70% sequence identity to the polypeptides encoded by plant UDP-Glc dehydrogenase or the relevant portion, preferably at least 80% identity to the polypeptides encoded by plant UDP-Glc dehydrogenase, and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptides encoded by plant UDP-Glc dehydrogenase and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptides encoded by plant UDP-Glc dehydrogenase and also include portions of such polypeptides.
  • the plant UDP-Glc dehydrogenase proteins of the present invention include the specifically identified and characterized variant herein described as well as allelic variants, conservative substitution variants and homologues that can be isolated/generated and characterized without undue experimentation following the methods outlined below.
  • the invention also relates to amino acid sequences coding for isolated soybean polypeptides of plant
  • the polypeptides of the invention include those which differ from the exemplified plant UDP-Glc dehydrogenase as a result of conservative variations.
  • an isolated plant UDP-Glc dehydrogenase protein can be a full-length plant UDP-Glc dehydrogenase protein or any homologue of such a protein, such as a plant UDP- Glc dehydrogenase protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycosylphosphatidyl inositol), wherein modified protein retains the physiological characteristics of natural plant UDP-Glc dehydrogenase.
  • a plant UDP-Glc dehydrogenase protein in which amino acids have been deleted e.g., a truncated version of the protein, such as a peptide
  • a homologue of a plant UDP-Glc dehydrogenase protein is a protein having an amino acid sequence that is sufficiently similar to a natural plant UDP-Glc dehydrogenase amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) a nucleic acid sequence encoding the natural plant UDP-Glc dehydrogenase amino acid sequence. Appropriate stringency requirements are discussed above.
  • Plant UDP-Glc dehydrogenase protein homologues can be the result of allelic variation of a natural gene encoding a plant UDP-Glc dehydrogenase protein. Natural genes are also referred to as "wildtype genes.” A natural, or wildtype, gene refers to the form of the gene found most often in nature. Plant UDP-Glc dehydrogenase protein homologues can be produced using techniques known in the art including, but not limited to, direct modifications to a gene encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • a "functional equivalent" of the plant UDP-Glc dehydrogenase protein is a protein which possesses a biological activity or immuno logical characteristic substantially similar to a biological activity or immunological characteristic of non-recombinant, or natural, plant UDP-Glc dehydrogenase.
  • the term "functional equivalent” is intended to include the fragments, variants, analogues, homologues, or chemical derivatives of a molecule which possess the biological activity of plant UDP-Glc dehydrogenase proteins of the present invention.
  • Homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin , et al. Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J. Mol. Evol. 36: 290-300(1993), fully incorporated by reference) which are tailored for sequence similarity searching.
  • the approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff, matrix and filter are at the default settings.
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff, et al. Proc. Natl.
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • crop plant means any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production.
  • Applicants further teach methods of recognizing variations in the DNA sequence of the soybean UDP-Glc dehydrogenase.
  • One method involves the introduction of a nucleic acid molecule (also known as a probe) having a sequence complementary to the plant UDP-Glc dehydrogenase gene of the invention under sufficient hybridizing conditions, as would be understood by those in the art.
  • Another method of recognizing DNA sequence variation associated with plant UDP-Glc dehydrogenase is direct DNA sequence analysis by multiple methods well known in the art (Ott, 1991).
  • Another embodiment of the present invention involves the detection of DNA sequence variation in the plant UDP-Glc dehydrogenase gene as represented by different plant genera, species, strains, varieties or cultivars.
  • the plant UDP-Glc dehydrogenase gene used for the probe can be from any plant for which the plant UDP-Glc dehydrogenase gene has been determined.
  • a particularly good probe for dicotyledonous plants would be that coding for the UDP-Glc dehydrogenase gene of soybean, while a particularly good probe for a monocotyledonous plant would be that coding for the plant UDP-Glc dehydrogenase gene of maize.
  • the sequence will bind specifically to one allele of the UDP-Glc dehydrogenase gene, or a fragment thereof, and in another embodiment will bind to multiple alleles.
  • detection methods include the polymerase chain reaction, restriction fragment length polymorphism (RFLP) analysis and single stranded conformational analysis.
  • Diagnostic probes useful in such assays of the invention include antibodies to the UDP- Glc dehydrogenase gene.
  • the antibodies to the UDP-Glc dehydrogenase gene may be either monoclonal or polyclonal, produced using standard techniques well known in the art (See
  • UDP-Glc dehydrogenase sequence used to elicit these antibodies can be any of the UDP-Glc dehydrogenase nucleic acid sequence variants discussed above.
  • Antibodies are also produced from peptide sequences of the UDP-Glc dehydrogenase gene using standard techniques in the art (See Protocols in Immunology. John Wiley & Sons, 1994). Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can also be prepared.
  • Assays to detect or measure plant UDP-Glc dehydrogenase polypeptide in a biological sample with an antibody probe may be based on any available format. For instance, in immunoassays where plant UDP-Glc dehydrogenase polypeptides are the analyte, the test sample, typically a biological sample, is incubated with anti-plant UDP-Glc dehydrogenase antibodies under conditions that allow the formation of antigen-antibody complexes.
  • Various formats can be employed, such as "sandwich” assay where antibody bound to a solid support is incubated with the test sample; washed, incubated with a second, labeled antibody to the analyte; and the support is washed again.
  • Analyte is detected by determining if the second antibody is bound to the support.
  • a competitive format which can be either heterogeneous or homogeneous, a test sample is usually incubated with an antibody and a labeled competing antigen, either sequentially or simultaneously.
  • Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microproj ectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; and
  • Agrobacterium-mediated transformation see, e.g., U.S. Patent Nos. 5,405,765, 5,472,869, 5,538,877, 5,538,880, 5,550,318, 5,641,664, 5,736,369 and 5,736369; Watson et al, Recombinant DNA. Scientific American Books (1992); Hinchee et al, Bio/Tech. 6:915-922 (1988); McCabe et ⁇ /., Bio/lech, 6:923-926 (1988); Torivama et ⁇ /.. Bio/Tech. 6:1072-1074 (1988); Fromm et al, Bio/Tech. 8:833-839 (1990); Mullins et al, Bio/Tech. 8:833-839 (1990); and, Raineri et al, Bio/Tech. 8:33-38 (1990)).
  • a preferred method of introducing the nucleic acid segments into plant cells is to infect a plant cell, an explant, a meristem or a seed with Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes transformed with the segment. While the wild-type
  • Agrobacterium rhizogenes may be used, the Agrobacterium tumefaciens should be "disarmed,” i.e., have its tumor-inducing activity removed, prior to use.
  • Preferred Agrobacterium tumefaciens strains include LBA4404, as described by Hoekema et al. (1983) Nature, 303:179-180, and EHA101 as described by Hood et al. (1986) J. Bacteriol., 168:1291-1301.
  • a preferred Agrobacterium rhizogenes strain is 15834, as described by Birot et al. (1987) Plant
  • the transformed plant cells are grown to form shoots, roots, and develop further into plants.
  • the nucleic acid segments can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of Agrobacterium tumefaciens.
  • the Ti plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Horsch et al, (1984)
  • Ti plasmids contain two regions essential for the production of transformed cells. One of these, named transfer DNA (T DNA), induces tumor formation. The other, termed virulent region, is essential for the introduction of the T DNA into plants.
  • T DNA transfer DNA
  • the transfer DNA region which transfers to the plant genome, can be increased in size by the insertion of the foreign nucleic acid sequence without its transferring ability being affected. By removing the tumor-causing genes so that they no longer interfere, the modified Ti plasmid can then be used as a vector for the transfer of the gene constructs of the invention into an appropriate plant cell, such being a "disabled Ti vector".
  • Method (1) requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • Method (2) requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
  • Method (3) requires regeneration or micropropagation.
  • T-DNA containing plasmid In the binary system, to have infection, two plasmids are needed: a T-DNA containing plasmid and a vir plasmid. Any one of a number of T-DNA containing plasmids can be used, the only requirement is that one be able to select independently for each of the two plasmids.
  • those plant cells or plants transformed by the Ti plasmid so that the desired DNA segment is integrated can be selected by an appropriate phenotypic marker.
  • phenotypic markers include, but are not limited to, antibiotic resistance, herbicide resistance or visual observation. Other phenotypic markers are known in the art and may be used in this invention.
  • transgenote refers to the immediate product of the transformation process and to resultant whole transgenic plants.
  • regeneration means growing a whole plant from a plant cell, a group of plant cells, a plant part or a plant piece (e.g., from a protoplast, callus, or tissue part). Plant regeneration from cultural protoplasts is described in Evans et al., "Protoplast
  • Parts obtained from the regenerated plant such as flowers, pods, seeds, leaves, branches, fruit, and the like are covered by the invention, provided that these parts comprise cells which have been so transformed. Progeny and variants, and mutants of the regenerated plants are also included within the scope of this invention, provided that these parts comprise the introduced
  • a “pod”, as used herein, is an expanded ovary composed of one carpel (the seeds are attached to the carpel).
  • a “seed”, as used herein, consists of an embryo contained within a maternally-derived seed coat (the testa). During early stages of seed development in pea, the seed also contains a triploid endosperm.
  • An “embryo”, as used herein, is made up of one (monocots) or two (dicots) cotyledons which make up the bulk of the embryo, a plumule, a radicle, a basal cell, and a suspensor cell.
  • a “cotyledon”, as used herein, is the first embryo seed leaf.
  • Genes successfully introduced into plants using recombinant DNA methodologies include, but are not limited to, those coding for the following traits: seed storage proteins, including modified 7S legume seed storage proteins (U.S. Patent Nos. 5,508,468, 5,559,223 and 5,576,203); herbicide tolerance or resistance (U.S. Patent Nos. 5,498,544 and 5,554,798; Powell et al, Science 232:738-743 (1986); Kaniewski et al, Bio/Tech. 8:750-754 (1990); Day et al, Proc. Natl. Acad. Sci. USA 88:6721-6725 (1991)); phytase (U.S. Patent No.
  • expression units or expression vectors or systems
  • an exogenously supplied nucleic acid sequence such as the sequence coding for UDP-Glc dehydrogenase protein in a plant.
  • Methods for generating expression units/systems/vectors for use in plants are well known in the art and can readily be adapted for use in expressing the UDP-Glc dehydrogenase protein in a plant cell.
  • a skilled artisan can readily use any appropriate plant/vector/expression system in the present methods following the outline provided herein.
  • the expression control elements used to regulate the expression of the protein can either be the expression control element that is normally found associated with the coding sequence (homologous expression element) or can be a heterologous expression control element.
  • Transcription initiation regions can include any of the various opine initiation regions, such as octopine, mannopine, nopaline and the like that are found in the Ti plasmids of Agrobacterium tumafacians.
  • plant viral promoters can also be used, such as the cauliflower mosaic virus 35S promoter to control gene expression in a plant.
  • plant promoters such as prolifera promoter, fruit-specific promoters, Ap3 promoter, heat shock promoters, seed-specific promoters, etc. can also be used.
  • the most preferred promoters will be most active in seedlings. Either a constitutive promoter (such as the CaMV or Nos promoter), an organ-specific promoter (such as the E8 promoter from tomato) or an inducible promoter is typically ligated to the protein or antisense encoding region using standard techniques known in the art.
  • the expression unit may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.
  • the expression units will typically contain, in addition to the protein sequence, a plant promoter region, a transcription initiation site and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the expression unit are typically included to allow for easy insertion into a preexisting vector.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct.
  • Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al, EMBO J3: 835-846 (1984)) or the nopaline synthase signal (Depicker et al, Mol. and Appl. Genet 1: 561-573 (1982)).
  • the resulting expression unit is ligated into or otherwise constructed to be included in a vector which is appropriate for higher plant transformation.
  • the vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic.
  • Replication sequences, of bacterial or viral origin are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range prokaryotic origin of replication is included.
  • a selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
  • DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobacterium transformations, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
  • the plant UDP-Glc dehydrogenase sequences of the present invention can also be fused to various other nucleic acid molecules such as Expressed Sequence Tags (ESTs), epitopes or fluorescent protein markers.
  • ESTs Expressed Sequence Tags
  • epitopes or fluorescent protein markers.
  • ESTs are gene fragments, typically 300 to 400 nucleotides in length, sequenced from the 3' or 5' end of complementary-DNA (cDNA) clones. Nearly 30,000 Arabidopsis thaliana ESTs have been produced by a French and an American consortium (Delseny et al., FEBS Lett.
  • GFP-fusion proteins have been used to localize and characterize a number of Arabidopsis genes, including geranylgeranyl pyrophosphate (GGPP)
  • An example of an effective disabling modification would be a single nucleotide deletion occurring at the beginning of a gene that would produce a translational reading frameshift. Such a frameshift would disable the gene, resulting in non-expressible gene product and thereby disrupting functional protein production by that gene. Protease production by the gene could be disrupted if the regulatory regions or the coding regions of the protease genes are disrupted.
  • disabling modifications would also be possible by other techniques including insertions, substitutions, inversions or transversions of nucleotides within the gene's DNA that would effectively prevent the formation of the protein coded for by the DNA.
  • antisense in which the function of a target gene in a cell or organism is blocked, by transfection of DNA, preferably an oligonucleotide, encoding antisense RNA which acts specifically to inhibit expression of the particular target gene.
  • the sequence of the antisense DNA is designed to result in a full or preferably partial antisense RNA transcript which is substantially complementary to a segment of the gene or mRNA which it is intended to inhibit.
  • the complementarity must be sufficient so that the antisense RNA can hybridize to the target gene (or mRNA) and inhibit the target gene's function, regardless of whether the action is at the level of splicing, transcription or translation.
  • the degree of inhibition readily discernible by one of ordinary skill in the art without undue experimentation, must be sufficient to inhibit, or render the cell incapable of expressing, the target gene.
  • the antisense RNA approach is but one of a number of known mechanisms which can be employed to block specific gene expression.
  • antisense an RNA sequence, as well as a DNA sequence coding therefor which is sufficiently complementary to a particular mRNA molecule for which the antisense RNA is specific to cause molecular hybridization between the antisense RNA and the mRNA such that translation of the mRNA is inhibited. Such hybridization must occur under in vivo conditions, that is, inside the cell. The action of the antisense RNA results in specific inhibition of gene expression in the cell. See Albers, et al, "Molecular Biology Of The Cell", 2nd Ed., Garland Publishing, Inc., New York, NY (1989), in particular, pages 195-196.
  • the antisense RNA of the present invention may be hybridizable to any of several portions of a target mRNA, including the coding sequence, a 3' or 5' untranslated region, or other intronic sequences.
  • a preferred antisense RNA is that complementary to UDP-glucose dehydrogenase mRNA.
  • the minimal amount of homology required by the present invention is that sufficient to result in hybridization to the specific target mRNA and inhibition of its translation or function while not affecting function of other mRNA molecules and the expression of other genes.
  • Antisense RNA is delivered to a cell by transformation or transfection with a vector into which has been placed DNA encoding the antisense RNA with the appropriate regulatory sequences, including a promoter, to result in expression of the antisense RNA in a host cell. It should be noted that to achieve down regulation of gene expression in plants the introduced (i.e., exogenous) transcript need not be "full-length" (e.g., see Zhong et al. (1992)
  • the introduced transcript will usually be at least 50 bases in length, more often at least 100 bases, even more often at least 200 bases and may be 500 or more bases in length.
  • the length of the exogenous sense transcript can also be expressed as a percentage of the length of the endogenous transcript.
  • the exogenous transcript will typically be at least about 5% as long as the endogenous transcript, more often at least about 10% as long, still more often at least about 20% as long, even more often at least about 50% as long.
  • Successful sense suppression also does not require that the introduced transcript comprise a sequence identical to that of the target (i.e., endogenous) gene. Rather, an introduced transcript with substantial identity (or substantial similarity) to the endogenous gene is sufficient.
  • the introduced transcript will have at least 60% nucleotide identity, more often at least 80% identity, still more often at least 85% identity, even more often at least 90% identity, still more often at least 95% identity, and most often at least 98% identity.
  • Recombinant DNA techniques allow plant researchers to circumvent these limitations by enabling plant geneticists to identify and clone specific genes for desirable traits, such as resistance to an insect pest, and to introduce these genes into already useful varieties of plants. Once the foreign genes have been introduced into a plant, that plant can than be used in conventional plant breeding schemes (e.g., pedigree breeding, single-seed-descent breeding schemes, reciprocal recurrent selection) to produce progeny which also contain the gene of interest.
  • conventional plant breeding schemes e.g., pedigree breeding, single-seed-descent breeding schemes, reciprocal recurrent selection
  • Genes can be introduced in a site directed fashion using homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be co ⁇ ected, and/or novel alterations may be engineered into the genome.
  • Open-Pollinated Populations The improvement of open-pollinated populations of such crops as rye, many maizes and sugar beets, herbage grasses, legumes such as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil palm and some rubber, depends essentially upon changing gene-frequencies towards fixation of favorable alleles while maintaining a high (but far from maximal) degree of heterozygosity. Uniformity in such populations is impossible and trueness-to-type in an open-pollinated variety is a statistical feature of the population as a whole, not a characteristic of individual plants.
  • the heterogeneity of open-pollinated populations contrasts with the homogeneity (or virtually so) of inbred lines, clones and hybrids.
  • Population improvement methods fall naturally into two groups, those based on purely phenotypic selection, normally called mass selection, and those based on selection with progeny testing.
  • Interpopulation improvement utilizes the concept of open breeding populations; allowing genes for flow from one population to another. Plants in one population (cultivar, strain, ecotype, or any germplasm source) are crossed either naturally (e.g., by wind) or by hand or by bees (commonly Apis mellifera L. or Megachile rotundata F.) with plants from other populations Selection is applied to improve one (or sometimes both) population(s) by isolating plants with desirable traits from both sources.
  • Mass Selection In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and their is no control over pollination, mass selection amounts to a form of random mating with selection. As stated above, the purpose of mass selection is to increase the proportion of superior genotypes in the population.
  • Synthetics A synthetic variety is produced by crossing inter se a number of genotypes selected for good combining ability in all possible hybrid combinations, with subsequent maintenance of the variety by open pollination. Whether parents are (more or less inbred) seed-propagated lines, as in some sugar beet and beans (Vicia) or clones, as in herbage grasses, clovers and alfalfa, makes no difference in principle. Parents are selected on general combining ability, sometimes by test crosses or topcrosses, more generally by polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfing or sib crossing). However, even if the parents are not deliberately inbred, selection within lines during line maintenance will ensure that some inbreeding occurs. Clonal parents will, of course, remain unchanged and highly heterozygous.
  • Broad based synthetics formed from 100 or more clones would be expected to be more stable during seed multiplication than narrow based synthetics.
  • Hybrids A hybrid is an individual plant resulting from a cross between parents of differing genotypes. Commercial hybrids are now used extensively in many crops, including corn (maize), sorghum, sugarbeet, sunflower and broccoli. Hybrids can be formed a number of different ways, including by crossing two parents directly (single cross hybrids), by crossing a single cross hybrid with another parent (three-way or triple cross hybrids), or by crossing two different hybrids (four-way or double cross hybrids). Strictly speaking, most individuals in an outbreeding (i.e., open-pollinated) population are hybrids, but the term is usually reserved for cases in which the parents are individuals whose genomes are sufficiently distinct for them to be recognized as different species or subspecies.
  • Hybrids may be fertile or sterile depending on qualitative and/or quantitative differences in the genomes of the two parents.
  • Heterosis, or hybrid vigor is usually associated with increased heterozygosity which results in increased vigor of growth, survival, and fertility of hybrids as compared with the parental lines which were used to form the hybrid.
  • Maximum heterosis is usually achieved by crossing two genetically different, highly inbred lines.
  • hybrids The production of hybrids is a well-developed industry, involving the isolated production of both the parental lines and the hybrids which result from crossing those lines.
  • hybrid production process see, e.g., Wright, Commercial Hybrid Seed Production 8:161-176. In Hybridization of Corp Plants, supra.
  • a cDNA library was synthesized from 5 ⁇ g of mRNA using the ⁇ -uni-Zap kit (Stratagene). About 30% of the ligated phase cDNA was packaged into ⁇ phages with the Gigagold system (Stratagene), producing more than 10 7 plaque- forming units in the primary library. Half of this library was amplified once on large Petri dishes and used for this study.
  • plaque-forming units per 145-mm dish were grown for 4 h at 42 °C. Plates were overlaid with a nitrocellulose filter, impregnated with 0.2 M isopropyl-thio- ⁇ - galactoside, and further incubated for 5 h at 37 °C. The nitrocellulose filters were removed and washed extensively in TBS + Tween 20 to remove cell debris. After blocking the membrane in TBS + Tween 20 +3% BSA, the filters were incubated with the p47phox antiserum in a 1 :3000 dilution for 1 h and washed with TBS - Tween 20 four times for 5 min.
  • Both strands were sequenced from various subclones using standard primers and synthesized oligonucleotide primers. Analysis of the cDNA sequence was performed with the Blast program tool (Atschul et al., 1990) and on the local computer system using the clone and Align programs (Scientific and Educational Software, Soft Line, PA).
  • a full-length cDNA insert was cloned into the pQE31 expression vector (Qiagen, Chatsworth, CA) and transformed into Escherichia coli XL-1. Expression of the His-tagged fusion protein was carried out in 100-mL scale by induction of the bacteria with 1 mM isopropylthio- ⁇ -galactoside for 5 h at 30°C. The fusion protein was purified under denaturing conditions on nitrilotriacetic acid-agarose according to the Qiagen protocol. After extensive dialysis of the purified protein against 10 mM sodium phosphate buffer (pH 7.4), the enzyme was used to immunize two rabbits (white New Zealand, Thomae Pharma, Biberach, Germany).
  • Antibodies were collected from a bleeding rabbit l i d after the second boost injection (750 ⁇ g of protein per animal and boost). IgGs were purified from the antiserum using a ProteinA column (Hitrap, Pharmacia) and used throughout this study.
  • RNA (10 ⁇ g per lane) was separated on a 1.1% formaldehyde agarose gel and transferred onto a nylon membrane (Hybond N + , Amersham) via downward capillary blotting. Hybridization was carried out according to the protocol of Church and Gilbert (1984) with a random primed probe (Ready To Go system, Pharmacia) of the soybean cDNA for UDP-GlcDH.
  • the blot was rehybridized with HI, a cDNA probe from bean, believed to be constitutively expressed (Wingate et al., 1988). (See Figure 3).
  • Example 5 Immunoprecipitation Soybean cell-suspension cultures were homogenized in 50 mM potassium-phosphate buffer (pH 7.5) containing 2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, and 0.5% (w/v) polyvinylpolypyrrolidone. The protein extract was fractionated by (NH 4 ) 2 SO 4 precipitation. The pellet corresponding to the 25 to 50% saturation fraction was desalted on a PD10 column
  • the IgG fraction was diluted from 1:80 to 1:1200 into the protein extract and incubated on ice for 3 h. Precipitated protein was collected by 15 min of centrifugation at 4°C. The pellet was washed once, resuspended in assay buffer, and used for the enzyme assay.
  • Example 6 Enzyme Assay
  • UDP-GLcDH was measured spectroscopically in a 1-mL assay adapted from Roberts and Cetorelli (1973) with some modifications.
  • the assay consists of 20 mM Tris-Cl (pH 8), ImM EDTA, 1 mM NAD + , 0.4 mM UDP-Glc, and 1 mM NaN 3 .
  • the reduction of NADH was measured as an increase at 340 nm. Controls were performed without NAD or UDP-Glc, showing a slight decrease at 340 nm, and then were subtracted.
  • a library was constructed from soybean cell culture mRNA. Poly(A) + RNA was isolated from 4-d-old cell culture (mid-log-phase) and used for the synthesis of a directional cDNA expression library. Of 5 x 10 5 plaques screened with the p47phox antiserum, 10 immune positive phages were identified and further purified. All of the positive clones cross-hybridized under stringent conditions, indicating the same cDNA for all identified clones.
  • the longest cDNA insert was sequenced and it contained a putative full-length clone of 1.7 kb.
  • the open reading frame of 1440 bases encodes a protein of 480 amino acids, and the predicted molecular mass is 52.9 kD. This size is very close to the observed molecular mass of the protein western blots.
  • the enzyme assay was linear over at least 1 h. Controls lacking UDP-Glc as a substrate did not show any increase in reduced NADH. A boiled enzyme control was also totally inactive.
  • the antibodies were used to immunoprecipitate UDP-GlcDH from the crude protein extract. A serial dilution of the IgG fraction was added to the protein extract from soybean and incubated on ice for several hours. After centrifugation, enzyme activity for UDP-GlcDH was measured in the supernatant and in the resuspended pellet. The enzyme activity can be completely precipitated by the polyclonal antibody. A fraction of this activity is measurable in the pellet. The enzyme in the precipitate is partially inhibited by the antibodies, presumably by direct interaction with epitopes significant for enzymatic activity.
  • the fusion protein could only be purified under denaturing conditions. Attempts to renature the enzyme have as yet been unsuccessful.
  • Example 10 Expression of the Enzyme during Plant Development
  • the expression of UDP-GlcDH was analyzed at the mRNA level by Northern blot hybridization. Total RNA was separated on an agarose gel and transfe ⁇ ed to a nylon membrane. Northern blot hybridization with a 32 P-labeled probe showed a high level expression in root tips and lateral roots and a moderate level of expression in the epicotyl and in expanding leaves (Figure 3). In contrast, the level of expression in the upper part of the main root, in the hypocotyl, and in mature leaves was much lower.
  • Example 11 Genome Structure
  • Controls values for LER leaf elongation rate
  • LER is the average value of leaves 1 and 2 between days 12 and 14.
  • UDP-Glc dehydrogenase activity was assayed by grinding the entire shoot tissue of
  • Catharanthus roseus Physiologia Plantarum, 64, 111-117.

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Abstract

Cette invention concerne des acides nucléiques dérivés de plantes qui codent une déshydrogénase de glucose de diphosphate d'uridine. Cette invention concerne également des fragments de ces acides nucléiques, des vecteurs et des cellules hôtes contenant ces acides nucléiques, ainsi que des molécules antisens de ces acides nucléiques. Cette invention concerne en outre des procédés permettant de modifier la composition des parois cellulaires, lesquels consistent à introduire dans des cellules végétales les acides nucléiques codant la déshydrogénase de glucose de diphosphate d'uridine. Ces acides nucléiques peuvent être utilisés afin de produire des plantes transgéniques qui possèdent un plus grand taux d'allongement des feuilles, ces acides pouvant être utilisés afin de manipuler la croissance des plantes.
PCT/US1998/022964 1997-10-30 1998-10-30 Gene de deshydrogenase de glucose de disphosphate d'uridine, et role que joue ce gene dans la modification de la croissance et des caracteristiques des plantes WO1999023200A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6399859B1 (en) 1997-12-10 2002-06-04 Pioneer Hi-Bred International, Inc. Plant uridine diphosphate-glucose dehydrogenase genes, proteins, and uses thereof
CN115290774A (zh) * 2022-07-21 2022-11-04 重庆医科大学 尿苷二磷酸葡萄糖醛酸在制备用于检测肝癌试剂中的应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6399859B1 (en) 1997-12-10 2002-06-04 Pioneer Hi-Bred International, Inc. Plant uridine diphosphate-glucose dehydrogenase genes, proteins, and uses thereof
US7098381B2 (en) 1997-12-10 2006-08-29 Pioneer Hi-Bred International, Inc. Plant uridine diphosphate-glucose dehydrogenase genes, proteins, and uses thereof
CN115290774A (zh) * 2022-07-21 2022-11-04 重庆医科大学 尿苷二磷酸葡萄糖醛酸在制备用于检测肝癌试剂中的应用

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