WO2001004315A2 - Compositions de genes rin et leurs procedes d'utilisation - Google Patents

Compositions de genes rin et leurs procedes d'utilisation Download PDF

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WO2001004315A2
WO2001004315A2 PCT/US2000/019035 US0019035W WO0104315A2 WO 2001004315 A2 WO2001004315 A2 WO 2001004315A2 US 0019035 W US0019035 W US 0019035W WO 0104315 A2 WO0104315 A2 WO 0104315A2
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plant
nucleic acid
gene
acid sequence
rin
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PCT/US2000/019035
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WO2001004315A3 (fr
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James Giovannoni
Steven Tanksley
Julia Vrebalov
Veeraragavan Padmanabhan
Diane Ruezinsky
Ruth White
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The Texas A & M University System
Cornell Research Foundation, Inc.
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Priority to AU59313/00A priority Critical patent/AU5931300A/en
Publication of WO2001004315A2 publication Critical patent/WO2001004315A2/fr
Publication of WO2001004315A3 publication Critical patent/WO2001004315A3/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
    • 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
    • C12N15/8249—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 ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8235—Fruit-specific
    • 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
    • C12N15/825—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 pigment 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
    • C12N15/8291—Hormone-influenced development

Definitions

  • the present invention relates generally to the RIN and MC genes. More specifically, it relates to methods and compositions for the modification of plant phenotypes with the RIN and MC genes.
  • the ripe phenotype is the summation of biochemical and physiological changes occurring at the terminal stage of fruit development rendering the organ edible and desirable to seed dispersing animals and valuable as an agricultural commodity. These changes, although variable among species, generally include modification of cell wall ultrastructure and texture, conversion of starch to sugars, increased susceptibility to post-harvest pathogens, alterations in pigment biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Rhodes, 1980; Hobson and Grierson, 1993). Several of theses ripening attributes translate to decreased shelf-life and high input harvest, shipping and storage practices, particularly via changes in firmness and the overall decrease in resistance to microbial infection of ripe fruit.
  • Climacteric fruit such as tomato, cucurbits, avocado, banana, peaches, plums, and apples
  • non-climacteric fruits such as strawberry, grape and citrus
  • Ethylene has been shown to be necessary for the coordination and completion of ripening in climacteric fruit via analysis of inhibitors of ethylene biosynthesis and perception (Yang, 1985; Tucker and Brady, 1987), in transgenic plants blocked in ethylene biosynthesis (Klee et al, 1991; Oeller et al., 1991 ; Picton et al., 1993a), and through examination of the None-ripe (Nr) ethylene perception mutant of tomato (Lanahan et al., 1994).
  • ripening genes including ACC synthase and ACC oxidase, have been shown via antisense gene repression to have profound influences on the onset and degree of ripening (Hamilton et al, 1990; Oeller et al, 1991). Although the sum effect of this research has been a wealth of information pertaining to the regulation of ethylene biosynthesis and its role in ripening, the molecular basis of developmental cues which initiate ripening- related ethylene biosynthesis, and additional aspects of ripening not directly influenced by ethylene, remain largely unknown (Theologis et al, 1993).
  • Nr mutation has been identified and represents a gene responsible for ethylene perception and/or signal transduction and is a tomato homologue of the Arabidopsis Ethylene response 1 (Etrl) gene (Yen et al, 1995; Wilkinson et al, 1995).
  • Tomato has served as a model for ripening of climacteric fruit.
  • Ripening-related genes have been isolated via differential gene expression patterns (Slater et al, 1985, Lincoln et al, 1987, Pear et al, 1989, Picton et al, 1993b) and biochemical function (DellaPenna et al, 1986; Sheehy et al, 1987; Ray et al, 1988; Biggs and Handa, 1989; Harriman and Handa,
  • Promoter analysis of ripening genes has been performed via examination of promoter/reporter construct activities in transient assay systems and transgenic plants. The result has been the identification of c s-acting promoter elements which are responsible for both ethylene and non-ethylene regulated aspects of ripening
  • ripening-related genes including polygalacturonase, pectinmethylesterase, ACC synthase, ACC oxidase, and phytoene synthase have been tested via antisense gene repression and/or mutant complementation in transgenic tomatoes.
  • the cell wall pectinase, polygalacturonase was shown to be necessary for ripening- related pectin depolymerization and pathogen susceptibility, however, the inhibition of PG expression had minimal effects on fruit softening (Smith et al, 1988, Giovannoni et al ., 1989, Kramer et al, 1990).
  • transgenic tomatoes inhibited in ethylene biosynthesis demonstrates that climacteric ripening represents a combination of both ethylene mediated and developmental control (Theologis et al, 1993).
  • antisense ACC synthase tomatoes which failed to produce ethylene did not ripen
  • gene expression analysis demonstrated that several ripening-related genes, including polygalacturonase and E8 are expressed in the absence of ethylene. This observation confirms the presence of a developmental (or non-ethylene regulated) component of ripening.
  • the invention provides an isolated nucleic acid sequence comprising a RIN gene.
  • the RIN gene may be further defined as isolatable from the nucleic acid sequence of SEQ ID NO:6, from SEQ ID NO:5 or alternatively from SEQ ID NO:8, or any combinations of the foregoing.
  • the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1172, about 25 to about 1172, about 30 to about 1172, about 40 to about 1172, about 60 to about 1172, about 100 to about 1172, about 200 to about 1172, about 400 to about 1172, about 600 to about 1172, about 800 to about 1172, or about 1000 to about 1172 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO:6 or SEQ ID NO:8.
  • the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:8.
  • An isolated nucleic acid in accordance with the invention may further comprise an enhancer element.
  • An exemplary enhancer comprises an intron.
  • Such isolated nucleic acids may also comprise a native or heterologous transcriptional terminator or enhancers, as desired.
  • the invention provides an expression vector comprising a RIN gene.
  • the expression vector may comprise a native or heterologous promoter operably linked to the RIN gene in either sense or antisense orientation relative to said promoter.
  • Any heterologous promoter may be used including a CaMV 35S, CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase, R-allele, root cell promoter, ⁇ -tubulin, ABA- inducible promoter, turgor-inducible promoter, rbcS, sucrose synthetase 1, alcohol dehydrogenase 1, pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase or S-E9 small subunit RuBP carboxylase promote
  • the expression vector may also comprise any selectable marker, for example, a selectable marker selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase.
  • the expression vector may be circular or may comprise a linear nucleic acid segment, such as in the case of an expression cassette.
  • the expression vector is a plasmid vector.
  • the expression vector may further include any other desired elements, such as an enhancer, a nucleic acid sequence encoding a transit peptide, or a heterologous terminator operably linked to said RIN gene, for example, a nos terminator.
  • the RIN gene included in the expression vector may include any of the RIN-gene containing compositions described herein and exemplified in the claims. Such compositions may comprise any number of contiguous nucleotides from, for example, SEQ ID NO: 6, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:8, as well as all possible combinations thereof.
  • the nucleic acid compositions also specifically include the open reading frames of the RIN cDNA sequences provided in SEQ ID NO:7, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:8.
  • the invention provides a transgenic plant comprising a stably transformed RIN-gene containing expression vector in accordance with the invention.
  • the plant may be any species of plant, for example, a tomato plant.
  • the transgenic plant is further defined as a fertile R 0 transgenic plant.
  • a seed of the fertile R 0 transgenic plant wherein said seed comprises said expression vector.
  • the transgenic plant is further defined as a progeny plant of any generation of a fertile Ro transgenic plant, wherein said R 0 transgenic plant comprises said expression vector.
  • a seed of the progeny plant wherein said seed comprises said expression vector.
  • the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a RIN gene; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant.
  • the invention also provides a seed of the crossed fertile transgenic plant, wherein said seed comprises said selected DNA.
  • the plant may be of any species, including a tomato plant.
  • the plant may also be inbred or hybrid.
  • the invention provides a method of manipulating the phenotype of a plant comprising the steps of: (i) obtaining an expression vector comprising a RIN gene in sense or antisense orientation; (ii) transforming a recipient plant cell with said expression vector; and (iii) regenerating a transgenic plant from said recipient plant cell, wherein the phenotype of said plant is altered based on the expression of said RIN gene in sense or antisense orientation.
  • Any method of transformation may be used, including microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation.
  • the step of transforming comprises Agrobacterium- mediated transformation and the plant is a tomato plant.
  • the invention provides a method of plant breeding comprising the steps of: (i) obtaining a transgenic plant comprising a selected DNA comprising a RIN gene; and (ii) crossing said transgenic plant with itself or a second plant.
  • said second plant is an inbred plant.
  • the method may further comprise the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant.
  • the second plant and said third plant are of the same genotype, and may be inbred plants.
  • the invention provides a transgenic plant cell stably transformed with a selected DNA comprising RIN gene.
  • the cell may be from any plant species including tomato.
  • the selected DNA may comprise any of the RIN-gene compositions described herein, for example, an expression vector as described above.
  • the invention provides a nucleic acid sequence comprising from about 17 to about 1138, about 25 to about 1138, about 30 to about 1138, about 40 to about 1138, about 60 to about 1138, about 100 to about 1138, about 200 to about 1138, about 400 to about 1138, about 600 to about 1138, about 800 to about 1138, or about 1000 to about 1138 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:3.
  • an isolated nucleic acid sequence is provided having the nucleic acid sequence of SEQ ID NO:3.
  • the invention provides an isolated nucleic acid sequence comprising a MC gene.
  • the MC gene may be further defined as isolatable from the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:7 or alternatively from SEQ ID NO:8, or any combinations of the foregoing.
  • the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1049, about 25 to about 1049, about 30 to about 1049, about 40 to about 1049, about 60 to about 1049, about 100 to about 1049, about 200 to about 1049, about 400 to about 1049, about 600 to about 1049, about 800 to about 1049, or about 1000 to about 1049, contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO:8.
  • the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
  • the invention provides an isolated nucleic acid sequence comprising from about 17 to about 1097, about 25 to about 1097, about 30 to about 1172, about 40 to about 1097, about 60 to about 1097, about 100 to about 1097, about 200 to about 1097, about 400 to about 1097, about 600 to about 1097, about 800 to about 1097, or about 1000 to about 1097 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:2.
  • the isolated nucleic acid sequence has the nucleic acid sequence of SEQ ID NO:2.
  • An isolated nucleic acid in accordance with the invention may further comprise an enhancer element.
  • An exemplary enhancer comprises an intron.
  • Such isolated nucleic acids may also comprise a native or heterologous transcriptional terminator or enhancers, as desired.
  • the invention provides an expression vector comprising a
  • the expression vector may comprise a native or heterologous promoter operably linked to the MC gene in either sense or antisense orientation relative to said promoter.
  • Any heterologous promoter may be used including a MC, CaMV 35S, CaMV 19S, nos, Adh, actin, histone, ribulose bisphosphate carboxylase, R-allele, root cell promoter, ⁇ -tubulin, ABA- inducible promoter, turgor-inducible promoter, rbcS, sucrose synthetase 1, alcohol dehydrogenase 1 , pea small subunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, CaMV 35s transcript, Potato patatin, actin, cab, PEPCase or S-E9 small subunit RuBP carboxylase promoter.
  • the expression vector may also comprise any selectable marker, for example, a selectable marker selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase.
  • the expression vector may be circular or may comprise a linear nucleic acid segment, such as in the case of an expression cassette.
  • the expression vector is a plasmid vector.
  • the expression vector may further include any other desired elements, such as an enhancer, a nucleic acid sequence encoding a transit peptide, or a heterologous terminator operably linked to said MC gene, for example, a nos terminator.
  • the MC gene included in the expression vector may include any of the MC-gene containing compositions described herein and exemplified in the claims. Such compositions may comprise any number of contiguous nucleotides from, for example, SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:8, as well as all possible combinations thereof.
  • the nucleic acid compositions also specifically include the open reading frames of the MC cDNA sequences provided in SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NO:8..
  • the invention provides a transgenic plant comprising a stably transformed MC-gene containing expression vector in accordance with the invention.
  • the plant may be any species of plant, for example, a tomato plant.
  • the transgenic plant is further defined as a fertile R 0 transgenic plant.
  • a seed of the fertile R 0 transgenic plant wherein said seed comprises said expression vector.
  • the transgenic plant is further defined as a progeny plant of any generation of a fertile R 0 transgenic plant, wherein said R 0 transgenic plant comprises said expression vector.
  • a seed of the progeny plant wherein said seed comprises said expression vector.
  • the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a MC gene; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant.
  • the invention also provides a seed of the crossed fertile transgenic plant, wherein said seed comprises said selected DNA.
  • the plant may be of any species, including a tomato plant.
  • the plant may also be inbred or hybrid.
  • the invention provides a method of manipulating the phenotype of a plant comprising the steps of: (i) obtaining an expression vector comprising a MC gene in sense or antisense orientation; (ii) transforming a recipient plant cell with said expression vector; and (iii) regenerating a transgenic plant from said recipient plant cell, wherein the phenotype of said plant is altered based on the expression of said MC gene in sense or antisense orientation.
  • Any method of transformation may be used, including microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation.
  • the step of transforming comprises Agrobacterium- mediated transformation and the plant is a tomato plant.
  • Still yet another aspect of the invention provides a method of plant breeding comprising the steps of: (i) obtaining a transgenic plant comprising a selected DNA comprising a MC gene; and (ii) crossing said transgenic plant with itself or a second plant.
  • said second plant is an inbred plant.
  • the method may further comprise the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant.
  • the second plant and said third plant are of the same genotype, and may be inbred plants.
  • Still yet another aspect of the invention provides a transgenic plant cell stably transformed with a selected DNA comprising MC gene.
  • the cell may be from any plant species including tomato.
  • the selected DNA may comprise any of the MC-gene compositions described herein, for example, an expression vector as described above.
  • the invention provides an MC promoter.
  • the promoter is defined as being isolatable from the nucleic acid sequence of SEQ ID NO: 8.
  • the MC promoter may comprise a nucleotide sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO: 8.
  • the promoter may be defined as comprising a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000 f SEQ ID NO:8, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201 to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO:8.
  • the MC promoter may also be further defined as comprising a contiguous stretch of about 12, about 17, about 25, about 30, about 40, about 60, about 80, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500 or about 2543 or longer segments of the nucleic acid sequence of SEQ ID NO: 8, including all intermediate lengths therein.
  • the contiguous nucleic acids may be further defined as from a nucleotide sequence comprised on the nucleic acid sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO:8.
  • the nucleic acid segment may be from a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201. to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO:8.
  • the invention provides MC promoter sequences included with additional elements, including an enhancer, a terminator, a selected coding region or any other desired sequences.
  • Such sequences may be provided as an expression vector, or may comprise an expression cassette isolated from the vector.
  • plants transformed with nucleic acids comprising an MC promoter as well as methods for making such plants.
  • FIG. 1 T-DNA constructs for delivery of sense or antisense RIN gene cDNA (CD- 43) sequences into plant genomes.
  • the base plasmid construct termed RIN-pBl 121 Sense/ Antisense (Kanamycin resistant -NPT II), was approximately 12.9 kb in length.
  • RB, LB Right and left T-DNA borders, respectively;
  • Nos-pro Nopaline synthase promoter driving expression of the NPTII gene;
  • NTPII Neomycin phosphotransferase (kanamycin resistance) gene; nos-ter: transcription termination sequence from the nopaline synthase gene;
  • Sma/EcoRV the resulting chimeric sequence is recognized by neither enzyme.
  • FIG. 2 T-DNA constructs for delivery of sense or antisense MC gene cDNA (CD-34) sequences into plant genomes.
  • the base plasmid construct termed Mc-pBI 121 Sense/ Antisense (Kanamicin resistance - NPTII), has an approximate size of 12.8 kb.
  • RB, LB right and left T-DNA borders, respectively; Nos-pro: nopaline synthase promoter driving expression of the NPTII gene; NPTII: neomycin phosphotransferase (kanamycin resistance) gene; HindUl, Sphl, Xbal, BamHI, Smal, EcoRI, Sad: DNA restriction endonucleases (enzymes); and Smal/EcoRV; SacI(T)/SmaI; SacI(F /EcoRV: resulting chimeric sequences are recognized by either enzyme.
  • FIG. 3 Manipulation of fruit ripening and carotenoid accumulation with the tomato RIN gene.
  • the top panel shows fruit from five independently transformed tomato lines (A-E) of the genotype rin/rin; mc/cm in the cultivar Ailsa Craig and transformed with RIN-pBl 121 Sense (FIG. 1). Primary transformants (TO) were confirmed for transgene integration via DNA gel-blot analysis and subsequently self-pollinated. Resulting seed were harvested and grown (TI generation) and analyzed for transgene segregation. Representative fully mature fruit from TI rin/rin; mc/mc individuals that harbor the sense RIN transgene (top) or have segregated it out (bottom) are shown.
  • transgene expression in the mutant background recovers the non-ripening phenotype and confers ripening.
  • Integration of the same transgene in multiple independent lines has a range of effects shown in A-E.
  • the bottom panel shows representative mature fruit from normal (RIN/RIN) and nearly isogenic mutant (rin/rin) Ailsa Craig tomato lines.
  • the fruit marked SS is a representative fruit from a normal (RIN/RIN; MC/MC) line also transformed with the RIN -pBH2l Sense construct but which shows inhibited ripening.
  • R ⁇ A gel-blot analysis indicated that fruit from this line was repressed in RIN gene expression suggestive of a sense-repression (co-suppression) effect. Note that while expression of the normal RIN transgene recovers ripening in mutant fruit, the large sepal phenotype remains (compare the sepals of A-E top to RIN/RIN).
  • FIG. 4 Manipulation of sepal development and inflorescence meristem determinacy with the MC gene.
  • An antisense MC gene construct (MC-pBI121, FIG. 2) was transferred into the normal tomato cultivar Ailsa Craig.
  • Primary transformants (TO) were confirmed for transgene integration via DNA gel-blot analysis and subsequently self-pollinated. Resulting seed were harvested and grown (TI generation) and analyzed for transgene segregation. Representative floral inflorescences from TI individuals and relevant controls are shown.
  • the left panel shows whole inflorescences (top, middle) and close-ups of sepals (bottom) from two independent TI lines (left and right respectively) which contain (+) or have segregated out (-) the MC-pBI121 antisense gene construct. Note that shoots (leaves) developed from the two (+) inflorescences (middle) but not from those which have segregated out the transgene (-, top).
  • the right panel shows the normal (Rin/Rin) and mutant (rin/rin) controls which have genotypes Rin/Rin; Mc/Mc and rin/rin; mc/mc, respectively.
  • the image labeled (S) is a representative mutant transgenic line (i.e., genotype rin/rin; mc/mc) into which a sense MC transgene has been introduced (MC-pBI121 Sense, FIG. 2) with relatively normal sized sepals and inflorescence determinacy (i.e., mutant mc phenotype recovered), yet which are undergoing premature and dramatic senescence.
  • FIG. 5 DNA gel-blot showing the Rin versus rin alleles as restriction fragment length polymorphisms (RFLP).
  • DNA was digested with restriction enzyme EcoRV, electrophoresed on a 1% agarose gel, transferred to a nylon membrane, and hybridized to radiolabeled CD-43 (RIN) full-length cDNA.
  • the membrane was washed and exposed to X-ray film to create the autoradiogram shown.
  • the lowest band corresponds to the Rin allele while the upper band corresponds to the mutant rin allele as shown by examination of the controls.
  • the normal (R) and mutant (r ) alleles are indicated above each lane and represent the corresponding genotype as determined by analysis of band segregation.
  • FIG. 6 Expression of the RIN (CD43) and MC (CD34) genes in normal and mutant fruit.
  • WT isogenic normal
  • Nr mutations Note the alteration in transcript size in the rin mutant due to the partial deletion/fusion of RIN and MC sequences as shown in FIG. 7.
  • FIG. 7 DNA sequence of the region of tomato chromosome 5 harboring the RIN and MC genes. Genomic DNA sequence is shown in the 5' - 3' orientation. Base 1 is the putative transcription start site (based on full-length cDNA sequence) of the RIN gene which ends at base 5,694. Bases 5695 through 8,237 separate the RIN from the MC transcribed regions. The putative transcription start site of the MC gene (based on full-length cDNA sequence) is 8,238 and transcription terminates at 13,830. No sequences upstream of the RIN gene transcribed region nor downstream of the MC transcribed region are shown. Coding sequence is in upper case while non-coding sequences including the RIN and MC gene introns are in lower case.
  • Non-transcribed sequences (5,695 - 8,237) are in lower case italics and underlined.
  • the RIN gene is comprised of 9 exons and 8 introns and the MC gene is comprised of 8 exons and 7 introns.
  • the rin mutation begins at a point within the 8 th intron of the RIN gene (i.e., between bases 4,993 - 5,404) and terminates within the first intron of the MC gene (/ ' . e. , between bases 8,440 - 10,594).
  • FIG. 8 DNA sequence of the RIN full-length cDNA (CD-43). The full cDNA sequence is shown in 5' - 3' orientation. Lower case letters refer to non-translated portions of the transcript while the upper case letters refer to the translated (coding) sequence.
  • FIG. 9 DNA sequence of the MC full-length cDNA (CD-34).
  • the full cDNA sequence is shown in 5' - 3' orientation.
  • Lower case letters refer to non-translated portions of the transcript while the upper case letters refer to the translated (coding) sequence.
  • the ripening of fleshy fruits represents a system of eukaryotic development unique to plants as well as an important component of agricultural quality and productivity. Greater understanding of the genetic and molecular basis of the ripening process will promote both our collective understanding of plant development and yield tools useful for sustaining and improving agricultural productivity and quality, while minimizing impact on the resources necessary for production.
  • the current invention provides such understanding by way of providing nucleic acids encoding the RIN and MC genes. By providing these sequences, the invention provides, for the first time, the ability to use genetic transformation techniques to manipulate a variety of plant characteristics which are associated with these genes in ways that cannot be accomplished via traditional breeding strategies including direct DNA transfer to species other than tomato.
  • CD-34 and CD-43 Two candidate cDNAs corresponding to the rin mutation were identified and termed CD-34 and CD-43. Both genes show altered transcript sizes in mutant versus normal fruit (FIG. 6). It was shown by the inventors through comparative sequencing of the normal versus mutant alleles of the rin locus (FIG. 7) that the rin mutation represents a deletion in parts of two adjacent genes (RIN and MC). DNA sequencing suggests that RIN (CD-43) and MC (CD- 34) are homologous and possess features consistent with function as transcription factors. Both genes are related to each other and are members of the MADS family of transcription factors (Theiben and Saedler, 1999). FIG. 7 depicts the DNA sequence of the adjacent RIN and MC loci with the deletion resulting in the rin mutation noted. FIG. 8 and FIG. 9 show the sequences of the RIN and MC cDNA sequences, respectively.
  • ripening occurs over a period of several days, depending on variety, and is characterized by softening, pectin solubilization, increased respiration and ethylene biosynthesis, enhanced pathogen susceptibility, heightened palatability, and accumulation of the characteristic red and orange carotenoid pigments lycopene and beta-carotene, respectively.
  • RIN as well as the NOR gene segregate as single traits, result in nearly complete inhibition of normal ripening as defined above, and their effects on ripening cannot be restored via application of exogenous ethylene (Tigchelaar et al, 1978).
  • the ripening phenotypes displayed by RIN and NOR demonstrate that the gene products encoded by the normal alleles at these loci are involved in the primary regulation of ripening (Hobson and Grierson, 1993; Giovannoni 1993; Gray et al., 1994). Because virtually nothing is known of the expression patterns or biochemical nature of the normal RIN and NOR gene products, the inventors initiated a genetic map-based cloning strategy for isolation of the corresponding normal alleles.
  • Tomato has served for decades as a model system for both plant genetics and fruit ripening, in part resulting in the availability of the tools for ripening gene isolation mentioned above.
  • Numerous mutations regulating various aspects of tomato fruit ripening have been identified over the years, most of which result in alteration of pigment biosynthesis and/or accumulation without significant effects on additional ripening characteristics (Rick, 1980; Grierson, 1986; Gray et al, 1994). Examples include the green ⁇ esh (gf; Ramirez and Tomes, 1964) and yellowflesh (r; Darby, 1978) mutants which inhibit ripening-related chlorophyll degradation and lycopene accumulation, respectively.
  • Tomato mutations exerting complete or nearly complete inhibition of overall ripening i.e. blocking changes not just in color but also texture, ethylene biosynthesis, pathogen susceptibility, flavor and aroma
  • homozygous rin and nor mutant fruit fail to display climacteric respiration and ethylene biosynthesis characteristic of normally ripening tomatoes (Tigchelaar et al, 1978), are highly resistant to microbial infection (Grierson, 1986), and are inhibited in their expression of ripening-related genes (DellaPenna et al, 1989; Picton et al, 1993). Although often referred to as recessive mutations, both rin and nor heterozygotes show significant effects on some ripening parameters, including reduced pathogen susceptibility and softening, resulting in extended shelf-life (Tigchelaar et al, 1978; Biggs and Handa, 1989).
  • Ripening is a unique and important plant process whose understanding has great significance in the agricultural arts. Isolation of genes regulating both the ethylene and non- ethylene mediated components of fruit ripening represents an important step in understanding the genetic basis of this complex developmental pathway. Although most research emphasis in recent years has been focused on elucidating the biosynthesis and function of ethylene during climacteric fruit ripening, the genetic and molecular basis of the developmental regulators which initiate ripening ethylene biosynthesis, and control the non-ethylene mediated ripening pathway, had remained a mystery.
  • the mutant phenotypes of the targeted RIN locus demonstrates that this gene is essential for normal ripening to occur and is a developmental regulator both of ethylene biosynthesis and non-ethylene regulated aspects of ripening.
  • the gene may be related to those involved in the regulation of other developmental programs. Insights gained into the ripening process as a result of the current invention will not only aid our understanding of overall plant development, but may enhance understanding of developmental processes in other eukaryotes as well.
  • ripening confers both positive and negative attributes to the resulting commodity. While ripening imparts desirable flavor, color, and texture, considerable expense and crop loss result as a consequence of negative ripening characteristics. For example, ripening related increases in fruit pathogen susceptibility is a major contributor to fruit loss both before and after harvest. This genetically regulated change in fruit physiology currently necessitates the use of pesticides, post-harvest fumigants, and controlled atmosphere storage and shipping mechanisms in attempts to minimize loss. In addition to being wasteful of energy and potentially harmful to the environment, such practices represent major expenses in fruit production.
  • the current inventors have isolated the ripening regulatory genes RIN and NOR, which allows for the first time the genetic enhancement through manipulation of said genes of positive ripening attributes and reduction of undesirable qualities in tomato and additional species.
  • the ability to improve fruit quality while reducing energy use, production costs, and environmental impact will promote the long term productivity and sustainability of commercial agriculture.
  • Tomato is the best available system for the map-based cloning of ripening genes because of the availability of: 1) single locus mutations inhibiting normal fruit ripening, 2) a high density RFLP map, 3) large populations segregating for targeted ripening loci, 4) a YAC library, and 5) established procedures for transformation and regeneration.
  • gene products have not been identified for either of the target genes, thus precluding immunological cloning strategies.
  • previous to the invention one could only speculate concerning patterns of normal RIN and NOR gene expression, thus exacerbating the already difficult task of identifying appropriate stages for differential screening strategies. Therefore, the current invention represents a major advance over the prior art, potentially allowing the creation of transgenic plants having greatly enhanced agronomic characteristics.
  • RIN and MC are primarily involved in fruit ripening and sepal development/ inflorescence determinacy, respectively. It was shown by the inventors that the RIN gene mutation (rin) greatly inhibits the ripening process with minimal effects on other plant tissues or even fruit prior to the onset of ripening. In addition, it is shown that the MC gene mutation (mc) results in much larger sepals than normal and loss of inflorescence meristem determinacy. Consequently, both the RIN and MC genes have potential for manipulation of fruit ripening and sepal development/ inflorescence determinacy, respectively.
  • Fruit ripening is a complex process ultimately rendering the fruit palatable and/or susceptible to biotic or abiotic process which result in seed liberation and dispersal. While specific ripening attributes vary among species the following general process are common to many fruits (see Seymour et al, 1993 for review), including tomato, and are all have been shown to be influenced by the RIN gene via characterization of the corresponding mutant:
  • Arabidopsis APETELA1 gene which is a member of the same gene family as RIN and MC, is known to influence inflorescence meristem determinacy in a manner similar to MC (Mandel et al, 1992). Furthermore, DNA sequence alignments suggest that MC is more closely related to API than any other plant gene currently in the public databases. The effects of API on Arabidopsis sepal development however are at the level of the decision to make a sepal versus a petal, while petal development in MC appears to be largely normal.
  • the rin mutation is currently widely used in tomato breeding for development of hybrid lines with slow-ripening/long-shelf-life characteristics.
  • the RIN gene could similarly be used for manipulation and control of ripening with potential for accelerated ripening of important early season crops, controlled or delayed ripening of crops permitting longer shipping handling, storage and post-retail shelf-life.
  • the fact that this gene has been cloned will permit its utilization in species other than tomato. Specific example of use include accelerated ripening of early season melons for favorable market position and pricing, and ripening control of bananas and strawberry, fruits which typically have short shelf-lives making shipping and handling more costly.
  • modified expression of the RIN gene in ripening fruits may be useful in elevating levels of important processing and nutritional compounds, such as antioxidant flavonoids and carotenoids in fruits and non-fruit tissues.
  • important processing and nutritional compounds such as antioxidant flavonoids and carotenoids
  • An example would be potential over-expression in maize seeds to enhance accumulation of antioxidant compounds for nutritional enhancement of the crop or for extraction.
  • RIN and MC gene orthologues may regulate maturation of seed pods (which are also carpels or "fruits", for example in soybean, pea, common bean) and/or cereal grains (e.g., rice, maize, wheat, sorghum).
  • seed pods which are also carpels or "fruits", for example in soybean, pea, common bean
  • cereal grains e.g., rice, maize, wheat, sorghum.
  • Protracted or accelerated maturation via manipulation of these genes may additionally impact quality characteristics such as total protein content, carbohydrate loading, nutritional composition (e.g., via altered levels of carotenoids such as beta-carotene and lycopene) and total yield.
  • FIG. 3 shows that delivery of the normal Rin allele into the genome of mutant plants results in a) conversion of fruit from unripe to ripe and b) results in a range of degrees of ripening and pigment accumulation.
  • the figure also shows that delivery of a sense RIN gene construct into the normal genetic background can result in inhibition of ripening.
  • the rin mutation is associated with the large sepal (macrocalyx or mc) phenotype in addition to a loss of determinacy in the inflorescence (floral) meristems.
  • the inventors have revealed that one gene (RIN) controls ripening, while the other (MC) controls sepal and inflorescence development. Note that in FIG. 3, while expression of the normal RIN transgene recovers ripening in mutant rin/rin fruit, the large sepal phenotype remains (compare the sepals of A-E top to RIN/RIN).
  • the inventors transferred an antisense MC gene (MC-pBI121 Antisense, FIG. 2) into normal tomatoes and have re-created the mc phenotype with respect to both sepal and inflorescence development by repression of the endogenous MC gene (FIG. 4).
  • MC-pBI121 Antisense FIG. 2
  • the sepals of flowers are generally thought to serve a protective purpose for developing flower and fruit tissues and may additionally serve as attractants for pollinating organisms. Examples of use of this gene contemplated by the inventors thus include: 1) manipulating sepal size in ornamental species and 2) manipulating sepal size in crops for the purpose of providing increased protection of developing fruits and flowers. Furthermore, manipulation of MC expression could be utilized to alter floral meristem determinacy resulting in plants with more or less fruits impacting yield and/or quality.
  • the RIN gene controls fruit senescence as demonstrated by the lack of senescence in tomato fruits harboring the rin mutation. Senescence or tissue death is thus clearly regulated by RIN in fruit and may be manipulated in non-fruit tissues via regulated expression of the RIN and/or related MC genes. Examples of use may include late fruit-ripening repression in banana or other tropical or sub-tropical fruits subject to rapid decay to permit desirable ripening but not advanced tissue damage reducing fruit quality and desirability. Over- expression in anthers may result in senescence yielding male-sterility, while if these genes are normally expressed in other senescing tissues, gene repression may be useful to inhibit senescence for example in vegetables (spinach, lettuce, cabbage, broccoli).
  • Fruit tissue from rin mutant tomato plants are highly resistant to infection by opportunistic microbial pathogens (Kramer et al, 1990).
  • Post-ripening repression (antisense or co-suppression) of the gene in tomato, or other species could thus be useful in inhibiting subsequent over-ripening and pathogen susceptibility of fruit.
  • activity of the RIN gene may participate in non-fruit pathogen resistance, for example, via repression of low-level of tissue or cell specific expression in response to pathogen attack. Consequently, RIN gene repression may have a positive impact on pathogen resistance in fruit and non-fruit tissues.
  • Specific examples of use might include 1) synchronized and controlled maturation of cereal grains via high level RIN expression late in seed development, 2) high level expression of MC later in the growing season to induce senescence and defoliation of cotton via over-expression in leaves prior to boll harvest, and 3) synchronized and accelerated or protracted maturation of seed pods via over-expression or repression, respectively of RIN in soybean.
  • stress responses such as responses to pathogen infection, and abiotic stress (temperature, water, mechanical damage) are mediated in part by ethylene
  • over-expression of the RIN and/or MC genes may positively impact the ability of plants to withstand biotic and abiotic insults, thus resulting in enhanced crop performance and yield.
  • the rin mutation is already used widely in breeding of fresh market and processing tomatoes.
  • Current phenotypic selection methods require confirmation of genotype at the rin locus through analysis of fruit or sepal development (i.e., later stages of plant development) with confirmation requiring analysis of subsequent progeny.
  • Such phenotypic screening requires considerable growth space and 2-3 months per plant generation cycle.
  • Isolation of the DNA sequences corresponding to the rin (and associated mc) mutation has permitted development of DNA markers based on sequence variation between the normal versus mutant tomato genotypes. Use of such markers allows for definitive genotyping of seedlings in a matter of 1 - 5 days.
  • An example of a DNA marker system based on the rin mutation is shown in FIG. 5.
  • RFLP restriction fragment length polymorphism
  • FIG. 7 depicts the D ⁇ A sequence of the adjacent RIN and MC loci with the deletion resulting in the rin mutation noted.
  • FIG. 8 and FIG. 9 show the sequences of the RIN and MC cD ⁇ A sequences, respectively.
  • an important advance of the instant invention is that it provides novel methods for the modification of plant phenotypes.
  • the invention allows the creation of plants with modified phenotypes.
  • the inventors specifically contemplate the use of the RIN and/or MC sequence, as well as all of the derivatives thereof which are provided by the invention, to genetically transform plant species for the purpose of altering plant phenotypes.
  • Exemplary phenotypic effects are those which are associated with fruit ripening or ethylene response.
  • RIN and/or MC sequences in accordance with the invention may be carried out using the native promoter, or alternatively, promoters that are inducible, viral, synthetic, constitutive as described (Poszkowski et al, 1989; Odell et al, 1985), and temporally regulated, spatially regulated (e.g., tissue-specific), and spatio-temporally regulated (Chau et al, 1989).
  • Types of effects which could be recognized on fruit ripening include, as described in detail above, processes related to changes in color, texture, flavor, aroma, shelf-life, ethylene responses, nutrient composition, cell wall metabolism, and susceptibility to pathogenesis associated with the ripening process.
  • effects on ethylene responsiveness which could be effected with the invention include either increased or decreased responsiveness to ethylene. Changes in response to ethylene may effect fruit ripening, organ abscission, seed or pollen dehiscence/shattering, tissue senescence, disease resistance, and response to environmental stresses including but not limited to drought, flooding, heat, cold, nutrient deficiency, high or low light intensity, mechanical damage and insect or pathogen infection. Modification of any of the foregoing effects in a plant, as well as any other effects associated with the RIN and/or MC gene, is specifically contemplated by the inventors and a part of the current invention.
  • any method employing the sequences described by the inventors may be used to realize the above-mentioned phenotypic effects in potentially any plant species, although fruiting effects can be expected to be realized only in species producing fruit.
  • fruit ripening or ethylene responsiveness could be decreased in a given plant by transformation of the plant with an expression vector comprising an antisense RIN gene.
  • an antisense MC gene could comprise the sequences provided herein, or could represent copies of homologous sequences from other plants isolated using the sequences of the invention. Decreases in fruit ripening, ethylene responsiveness or other fruit characteristics could alternatively be realized by use of co-suppression by way of introduction of additional
  • RIN and/or MC sequences into a host genome.
  • RIN and/or MC sequences preferably comprising a functional expression unit
  • cosuppression of any functional native and/or MC sequences could be realized, and thereby the phenotype of the plant be modified with respect to traits effected by RIN and/or MC.
  • the effect could be realized potentially by use of the RIN and/or MC promoters, coding sequences or terminators, or using heterologous versions thereof.
  • transgene comprising a sequence of the invention
  • that transgene may be passed to any subsequent generation by standard plant breeding protocols.
  • Such breeding can allow the transgene to be introduced into different lines, preferably of an elite agronomic background, or even to different species which can be made sexually compatible with the plant having the transgene. Breeding protocols may be aided by the use of genetic markers which are closely linked to the genes of interest.
  • the instant invention extends to any plant which has been directly introduced with a transgene prepared in accordance with the invention, or which has received the transgene by way of crossing with a plant having such a transgene.
  • the invention may additionally be applied to any plant species.
  • a plant prepared in accordance with the invention will be a fruiting plant, for example, tomato, berries such as strawberries and raspberries, banana, kiwi, avocado, melon, mango, papaya, lychee, pear, stoone fruits such as peach, apricot, plum and cherry, in addition to true (anatomical) fruits commonly referred to as "vegetables” including peppers, eggplant, okra, and other non-melon curcubuts such as cucumber and squash.
  • an expression vector containing a sense or antisense RIN and/or MC coding region and an appropriate selectable marker may be used in accordance with the invention, for example, use of Agrobacterium- mediated D ⁇ A transfer, microprojectile bombardment, direct D ⁇ A transfer into pollen, by injection of DNA into reproductive organs of a plant, or by direct injection of DNA into the cells of immature embryos followed by the rehydration of desiccated embryos, or by direct DNA uptake by protoplasted cells.
  • transgenic plants created in accordance with the current invention may be used for virtually any purpose deemed of value to the grower or to the consumer.
  • the fruit of tomato plants with enhanced fruit ripening characteristics may be harvested and sold to consumers or used in the production of various food products.
  • seed could be harvested from the fruit of a plant prepared in accordance with the instant invention, and the seed may be sold to farmers for planting in the field or may be directly used as food, either for animals or humans.
  • products may be made from the seed itself, for example, oil, starch, pharmaceuticals, and various industrial products. Such products may be made from particular plant parts or from the entire plant.
  • the inventors have identified the genomic DNA spanning the region between these coding sequence, which includes the 5' region of the MC gene, which is expected to correspond to the promoter region of this gene.
  • Such a promoter region may find use in agricultural biotechnology applications in view of the unique expression profile of the MC gene.
  • the MC gene is expressed primarily in sepals with much lower expression in fruit in normal tomato. As such, the MC promoter is a good candidate as a molecular tool for sepal, and to a lesser extent, fruit-specific gene expression.
  • sequences provided here likely contain all of the MC promoter sequences required for MC sepal and fruit-specific expression and would also be a tool by which any additional adjacent sequences could be recovered.
  • sequence upstream of MC leads into the RIN gene is strongly suggestive of the fact that all required upstream regulatory sequences for sepal and fruit- specific expression of MC axe contained in the sequence in FIG. 7, as additional regulatory sequences are not likely to extend into the adjacent gene, although this is possible.
  • one embodiment of the invention comprises a MC promoter.
  • the MC promoter may be defined as comprising a nucleotide sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO:8.
  • the promoter may be defined as comprising a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000 of SEQ ID NO:8, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201 to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO:8.
  • Such compositions specifically include the following sequences: nucleotide 7700 to nucleotide 8000 of SEQ ID NO:8, nucleotide 7800 to nucleotide 8100 of SEQ ID NO:8, nucleotide 7900 to nucleotide 8237 of SEQ ID NO:8, nucleotide 7800 to nucleotide 8000 of SEQ ID NO:8, nucleotide 7900 to nucleotide 8100 of SEQ ID NO:8, nucleotide 8000 to nucleotide 8237 of SEQ ID NO:8, nucleotide 8100 to nucleotide 8237 of SEQ ID NO:8, nucleotide 8150 to nucleotide 8237 of SEQ ID NO:8, and nucleotide
  • the MC promoter may be further defined as comprising a contiguous stretch of about 12, about 17, about 25, about 30, about 40, about 60, about 80, about 100, about 150, about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500 or about 2543 or longer segments of the nucleic acid sequence of SEQ ID NO:8, including all intermediate lengths therein.
  • the contiguous nucleic acids may be further defined as from a nucleotide sequence comprised on the nucleic acid sequence defined by nucleotide 5695 to nucleotide 8237 of SEQ ID NO:8.
  • the nucleic acid segment may be from a nucleotide sequence selected from the group consisting of nucleotide 5695 to nucleotide 6000 of SEQ ID NO:8, nucleotide 6001 to nucleotide 6400 of SEQ ID NO:8, nucleotide 6401 to nucleotide 6800 of SEQ ID NO:8, nucleotide 6801 to nucleotide 7200 of SEQ ID NO:8, nucleotide 7201 to nucleotide 7600 of SEQ ID NO:8, nucleotide 7601 to nucleotide 8000 of SEQ ID NO:8, or nucleotide 8001 to nucleotide 8237 of SEQ ID NO: 8.
  • the contiguous stretch of nucleic acids may be from a sequence selected from the group consisting of: from about nucleotide 7700 to about nucleotide 8237 of SEQ ID NO:8, from nucleotide 7700 to nucleotide 8000 of SEQ ID NO:8, from nucleotide 7800 to nucleotide 8100 of SEQ ID NO:8, from nucleotide 7900 to nucleotide 8237 of SEQ ID NO:8, from nucleotide 7800 to nucleotide 8000 of SEQ ID NO:8, from nucleotide 7900 to nucleotide 8100 of SEQ ID NO:8, from nucleotide 8000 to nucleotide 8237 of SEQ ID NO:8, from nucleotide 8100 to nucleotide 8237 of SEQ ID NO:8, from nucleotide 8150 to nucleotide 8237 of SEQ ID NO:8, and from nucleotide 8175 to nucleo
  • the invention also comprises the MC promoter sequences included with additional elements, including an enhancer, a terminator, a selected coding region in either sense or antisense orientation or any other desired sequences.
  • sequences may be provided as an expression vector, or may comprise an expression
  • the sequences provided herein also provide the molecular tools for identification of the RIN promoter.
  • This gene is expressed in ripening fruit with significant induction occurring just as the fruit begin to ripen (FIG. 6), and thus could potentially be used as a molecular tool for fruit and ripening-specific gene expression.
  • FOG. 6 ripening-specific gene expression.
  • the promoter region can readily be discerned from the coding region and isolated for use in the expression of selected genes.
  • the entire 5' and coding region of the RIN gene could be transformed into a selected plant.
  • the rin/rin; mc/mc mutation results in deletion of the entire intervening sequence between the RIN and MC genes, in addition to deletion of the last exon of the i?/N gene and the first exon of the MC gene (see FIG. 7).
  • the result is that in the mutant a fused gene is expressed which is comprised of the majority of the RIN gene (minus the last exon) fused to the majority of the MC gene (minus the first exon) (see FIG. 6).
  • This mutant fused gene product is considerably larger than the RIN or MC mRNAs, approximately 1.8 kb versus approximately 1.1 kb, and displays fruit and ripening-specific expression patterns very similar (basically identical) to the normal RIN gene (FIG. 6).
  • the mutant gene is expressed in fruit and initiated in expression during ripening. There is no expression in sepals (as is the case with the normal RIN gene).
  • the mutant demonstrates that a) sequences downstream of the RIN gene (i.e., between RIN and MC) are not important for RIN expression, and b) the sequences between RIN and MC (the putative MC promoter) are required, though not necessarily sufficient, for sepal-specific expression.
  • vectors which may be employed in conjunction with plant transformation techniques according to the invention will be known to those of skill of the art in light of the present disclosure (see, for example, Sambrook et al, 1989; Gelvin et al, 1990).
  • the techniques of the current invention are thus not limited to any particular nucleic acid sequences in conjunction with the RIN and/or MC nucleic acid sequences provided herein.
  • Exemplary sequences for use with the invention include those provided in SEQ ID NOs: 2, 3, 5, 6, 7, and 8.
  • sequences of the invention will be in the alteration of plant phenotypes by genetic transformation of plants with sense or antisense RIN and/or MC genes.
  • the RIN and/or MC genes may be provided with other sequences.
  • an expressible coding region that is not necessarily a marker coding region is employed in combination with a marker coding region, one may employ the separate coding regions on either the same or different DNA segments for transformation. In the latter case, the different vectors are delivered concurrently to recipient cells to maximize cotransformation.
  • the choice of any additional elements used in conjunction with the RIN and/or MC sequences will often depend on the purpose of the transformation.
  • One of the major purposes of transformation of crop plants is to add commercially desirable, agronomically important traits to the plant.
  • Such traits include, but are not limited to, processes related to changes in fruit color, texture, flavor, aroma, shelf-life, ethylene responses, nutrient composition, cell wall metabolism, susceptibility to pathogenesis associated with the ripening process, organ abscission, seed or pollen dehiscence/shattering, tissue senescence, disease resistance, and response to environmental stresses including but not limited to drought, flooding, heat, cold, nutrient deficiency, high or low light intensity, mechanical damage and insect or pathogen infection.
  • the present inventors contemplate the transformation of a recipient cell with more than transformation construct.
  • Two or more transgenes can be created in a single transformation event using either distinct selected-gene encoding vectors, or using a single vector incorporating two or more gene coding sequences.
  • the RIN and MC sequences provided by SEQ ID NOs: 6 and 7, could be transformed into a single plant.
  • the RIN and/or MC sequences could also be employed in conjunction with a NOR sequence.
  • viral vectors for plant transformation.
  • Such vectors include, for example, wheat dwarf virus (WDV) "shuttle" vectors, such as pWl-11 and PW1-GUS (Ugaki et al, 1991). These vectors are capable of autonomous replication in plant cells as well as E. coli, and as such may provide increased sensitivity for detecting DNA delivered to transgenic cells.
  • WDV wheat dwarf virus
  • a replicating vector also may be useful for delivery of genes flanked by DNA sequences from transposable elements such as Ac, Ds, or Mu.
  • transposable elements would be useful for introducing DNA fragments lacking elements necessary for selection and maintenance of the plasmid vector in bacteria, e.g., antibiotic resistance genes and origins of DNA replication. It also is proposed that use of a transposable element such as Ac, Ds, or Mu would actively promote integration of the desired DNA and hence increase the frequency of stably transformed cells.
  • Co-transformation may be achieved using a vector containing the marker and another gene or genes of interest.
  • different vectors e.g., plasmids
  • the plasmids may be concurrently delivered to the recipient cells.
  • Vectors used for plant transformation may include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning system.
  • vector or "expression vector”
  • all of the foregoing types of vectors, as well as nucleic acid sequences isolated therefrom, are included. It is contemplated that utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. Introduction of such sequences may be facilitated by use of bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes.
  • BACs for Agrobacterium-mediated transformation was disclosed by Hamilton et ⁇ l (1996).
  • DNA segments used for transforming plant cells will, of course, generally comprise the cDNA, gene or genes which one desires to introduced into and have expressed in the host cells. These DNA segments can further include, in addition to a i?/N and/or coding sequence, structures such as promoters, enhancers, polylinkers, or even regulatory genes as desired.
  • the DNA segment or gene chosen for cellular introduction will often encode a protein which will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and/or which will impart an improved phenotype to the resulting transgenic plant. However, this may not always be the case, and the present invention also encompasses transgenic plants incorporating non-expressed transgenes.
  • Preferred components likely to be included with vectors used in the current invention are as follows.
  • Preferred constructs will generally include a plant promoter such as the CaMV 35S promoter (Odell et al, 1985), or others such as CaMV 19S (Lawton et al, 1987), nos (Ebert et al, 1987), Adh (Walker et al, 1987), sucrose synthase (Yang & Russell, 1990), a-tubulin, actin (Wang et al, 1992), cab (Sullivan et al, 1989), PEPCase (Hudspeth & Grula, 1989) or those associated with the R gene complex (Chandler et al, 1989).
  • a plant promoter such as the CaMV 35S promoter (Odell et al, 1985), or others such as CaMV 19S (Lawton et al, 1987), nos (Ebert et al, 1987), Adh (Walker et al, 1987), sucrose synthase (Yang & Russell, 1990), a-tubulin, actin (Wang
  • Tissue specific promoters such as root cell promoters (Conkling et al, 1990) and tissue specific enhancers (Fromm et al, 1989) are also contemplated to be particularly useful, as are inducible promoters such as ABA- and turgor-inducible promoters.
  • leader sequences are contemplated to include those which include sequences predicted to direct optimum expression of the attached gene, i.e., to include a preferred consensus leader sequence which may increase or maintain mRNA stability and prevent inappropriate initiation of translation.
  • sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants, and in tomato in particular, will be most preferred.
  • vectors for use in accordance with the present invention may be constructed to include the ocs enhancer element.
  • This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of Agrobacterium (Ellis et al. , 1987), and is present in at least 10 other promoters (Bouchez et al, 1989). It is proposed that the use of an enhancer element, such as the ocs element and particularly multiple copies of the element, will act to increase the level of transcription from adjacent promoters when applied in the context of plant transformation.
  • RIN and/or MC coding sequences may be introduced under the control of novel promoters or enhancers, etc., or perhaps even homologous or tissue specific (e.g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters or control elements.
  • tissue specific e.g., root-, collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific
  • a particular use of the present invention will be the targeting sense or antisense RIN and/or MC expression in a tissue- specific manner. For example, these genes could be targeted to the fruit.
  • tissue-specific promoters for use in tissue-specific targeting of genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos and mas promoters which have higher activity in roots or wounded leaf tissue; a truncated (-90 to +8) 35S promoter which directs enhanced expression in roots, and an a-tubulin gene that directs expression in roots.
  • tissue specific expression may be functionally accomplished by introducing a constitutively expressed gene (all tissues) in combination with an antisense gene that is expressed only in those tissues where the gene product is not desired.
  • a gene coding for a RIN and/or MC sequence may be introduced such that it is expressed in all tissues using the 35S promoter from Cauliflower Mosaic Virus. Expression of an antisense transcript of the same RIN and/or MC gene in the fruit of a plant would prevent expression of the RIN and/or MC gene only in the fruit.
  • tissue-specific promoter sequences for use in accordance with the present invention.
  • one may first isolate cDNA clones from the tissue concerned and identify those clones which are expressed specifically in that tissue, for example, using Northern blotting.
  • tissue concerned a tissue concerned
  • identify those clones which are expressed specifically in that tissue for example, using Northern blotting.
  • the promoter and control elements of corresponding genomic clones may then be localized using the techniques of molecular biology known to those of skill in the art.
  • sense or antisense RIN and/or MC genes in transgenic plants may in some cases be desired only under specified conditions. It is contemplated that expression of such genes at high levels may have detrimental effects. It is known that a large number of genes exist that respond to the environment. For example, expression of some genes such as rbcS, encoding the small subunit of ribulose bisphosphate carboxylase, is regulated by light as mediated through phytochrome. Other genes are induced by secondary stimuli. A number of genes have been shown to be induced by ABA (Skriver and Mundy, 1990). Therefore, in particular embodiments, inducible expression of the nucleic acid sequences of the invention may be desired.
  • rbcS encoding the small subunit of ribulose bisphosphate carboxylase
  • a RIN and/or MC gene will be desired only in a certain time period during the development of the plant. Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of certain genes associated with fruit ripening will only be expressed at certain stages of fruit development.
  • vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This will generally be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed. Transit or signal peptides act by facilitating the transport of proteins through intracellular membranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes, whereas signal peptides direct proteins through the extracellular membrane.
  • intracellular membranes e.g., vacuole, vesicle, plastid and mitochondrial membranes
  • a particular example of such a use concerns the direction of a herbicide resistance selectable marker gene, such as the EPSPS gene, to a particular organelle such as the chloroplast rather than to the cytoplasm.
  • a herbicide resistance selectable marker gene such as the EPSPS gene
  • organelle such as the chloroplast rather than to the cytoplasm.
  • rbcS transit peptide which confers plastid-specific targeting of proteins.
  • Terminators may be useful to target DNA itself within a cell. For example, it may be useful to target introduced DNA to the nucleus as this may increase the frequency of transformation. Within the nucleus itself it would be useful to target a gene in order to achieve site specific integration. For example, it would be useful to have an gene introduced through transformation replace an existing gene in the cell. (ii) Terminators
  • Transformation constructs prepared in accordance with the invention will typically include a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the poly-adenylation of the mRNA produced by coding sequences operably linked to a NOR and/or MC gene.
  • the native RIN and/or MC gene is used.
  • a heterologous 3' end may enhance the expression of sense or antisense RIN and/or MC sequences.
  • Terminators which are deemed to be particularly useful in this context include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3' end) (Bevan et al, 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato.
  • Regulatory elements such as Adh intron (Callis et al, 1987), sucrose synthase intron (Vasil et al, 1989) or TMV omega element (Gallie et al, 1989), may further be included where desired.
  • Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and which facilitate the transport of the protein into or through intracellular or extracellular membranes, are termed transit (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, golgi apparatus and outside of the cellular membrane).
  • transit usually into vacuoles, vesicles, plastids and other intracellular organelles
  • signal sequences usually to the endoplasmic reticulum, golgi apparatus and outside of the cellular membrane.
  • translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the stability of the protein (U.S. Patent No. 5,545,818, incorporated herein by reference in its entirety).
  • vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic plant or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed.
  • Marker genes are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can "select” for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify through observation or testing, i.e., by "screening”' (e.g., the green fluorescent protein).
  • a selective agent e.g., a herbicide, antibiotic, or the like
  • screening e.g., the green fluorescent protein
  • selectable or screenable markers also are genes which encode a "secretable marker" whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which are secretable antigens that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity.
  • Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
  • small, diffusible proteins detectable e.g., by ELISA
  • small active enzymes detectable in extracellular solution e.g., ⁇ -amylase, ⁇ -lactamase, phosphinothricin acetyltransferase
  • proteins that are inserted or trapped in the cell wall e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S.
  • a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous.
  • a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter- leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies.
  • a normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.
  • neo Paneo
  • bar which confers bialaphos or phosphinothricin resistance
  • a mutant EPSP synthase protein Hidec et al, 1988
  • a mutant acetolactate synthase ALS
  • ALS acetolactate synthase
  • selectable marker capable of being used in systems to select transformants are those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes.
  • the enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al, 1986; Twell et al, 1989) causing rapid accumulation of ammonia and cell death.
  • Screenable Markers Screenable markers that may be employed include a ⁇ -glucuronidase (GUS) or uidA gene which encodes an enzyme for which various chromogenic substrates are known; an R- locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al, 1988); a ⁇ -lactamase gene (Sutcliffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al, 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols; an ⁇ -amylase gene (Ikuta et al, 1990); a tyrosinase gene (Katz et al, 1983) which encodes an enzyme capable of oxidizing tyrosine to DO
  • Genes from the maize R gene complex can also be used as screenable markers.
  • the R gene complex in maize encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue.
  • Maize strains can have one, or as many as four, R alleles which combine to regulate pigmentation in a developmental and tissue specific manner.
  • an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector.
  • a maize line carries dominant alleles for genes encoding for the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, Al, A2, Bzl and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation.
  • Exemplary lines include Wisconsin 22 which contains the rg-Stadler allele and TR112, a K55 derivative which is r-g, b, PI.
  • any genotype of maize can be utilized if the CI and R alleles are introduced together.
  • Another screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene.
  • the presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It also is envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
  • the gene which encodes green fluorescent protein (GFP) is contemplated as a particularly useful reporter gene (Sheen et al, 1995; Haseloff et al, 1997; Reichel et al, 1996; Tian et al, 1997; WO 97/41228). Expression of green fluorescent protein may be visualized in a cell or plant as fluorescence following illumination by particular wavelengths of light. Where use of a screenable marker gene such as lux or GFP is desired, the inventors contemplated that benefit may be realized by creating a gene fusion between the screenable marker gene and a selectable marker gene, for example, a GFP-NPTII gene fusion. This could allow, for example, selection of transformed cells followed by screening of transgenic plants or seeds.
  • a selectable marker gene for example, a GFP-NPTII gene fusion. This could allow, for example, selection of transformed cells followed by screening of transgenic plants or seeds.
  • Negative Selectable Markers Introduction of genes encoding traits that can be selected against may be useful for eliminating undesirable linked genes. It is contemplated that when two or more genes are introduced together by cotransformation that the genes will be linked together on the host chromosome. For example, a gene encoding Bt that confers insect resistance on the plant may be introduced into a plant together with a bar gene that is useful as a selectable marker and confers resistance to the herbicide Liberty® on the plant. However, it may not be desirable to have an insect resistant plant that also is resistant to the herbicide Liberty®. It is proposed that one also could introduce an antisense bar gene that is expressed in those tissues where one does not want expression of the bar gene, e.g., in whole plant parts. Hence, although the bar gene is expressed and is useful as a selectable marker, it is not useful to confer herbicide resistance on the whole plant. The bar antisense gene is a negative selectable marker.
  • a homologous recombinant may be identified through the inactivation of a gene that was previously expressed in that cell.
  • the antisense gene to neomycin phosphotransferase II (NPT II) has been investigated as a negative selectable marker in tobacco (Nicotiana tabacum) and Arabidopsis thaliana (Xiang. and Guerra, 1993).
  • NPT II neomycin phosphotransferase II
  • both sense and antisense NPT II genes are introduced into a plant through transformation and the resultant plants are sensitive to the antibiotic kanamycin.
  • negative selectable markers also may be useful in other ways.
  • One application is to construct transgenic lines in which one could select for transposition to unlinked sites.
  • the transposable element In the process of tagging it is most common for the transposable element to move to a genetically linked site on the same chromosome.
  • a selectable marker for recovery of rare plants in which transposition has occurred to an unlinked locus would be useful.
  • the enzyme cytosine deaminase may be useful for this purpose (Stouggard, 1993). In the presence of this enzyme the compound 5-fluorocytosine is converted to 5-fluorouracil which is toxic to plant and animal cells.
  • transposable element is linked to the gene for the enzyme cytosine deaminase
  • the parental plants and plants containing transpositions to linked sites will remain sensitive to 5- fluorocytosine.
  • Resistance to 5-fluorocytosine is due to loss of the cytosine deaminase gene through genetic segregation of the transposable element and the cytosine deaminase gene.
  • Other genes that encode proteins that render the plant sensitive to a certain compound will also be useful in this context.
  • T-DNA gene 2 from Agrobacterium tumefaciens encodes a protein that catalyzes the conversion of ⁇ -naphthalene acetamide (NAM) to ⁇ - naphthalene acetic acid (NAA) renders plant cells sensitive to high concentrations of NAM (Depicker et ⁇ /., 1988).
  • NAM ⁇ -naphthalene acetamide
  • NAA ⁇ - naphthalene acetic acid
  • negative selectable markers may be useful in the construction of transposon tagging lines. For example, by marking an autonomous transposable element such as Ac, Master Mu, or En Spn with a negative selectable marker, one could select for transformants in which the autonomous element is not stably integrated into the genome.
  • DNA may be introduced into plants for the purpose of expressing RNA transcripts that function to affect plant phenotype yet are not translated into protein.
  • Two examples are antisense RNA, which is discussed in detail below, and RNA with ribozyme activity. Both may serve possible functions in reducing or eliminating expression of native or introduced plant genes, for example, a RIN and/or MC gene.
  • DNA need not be expressed to effect the phenotype of a plant.
  • Genes also may be constructed or isolated, which when transcribed, produce RNA enzymes (ribozymes) which can act as endoribonucleases and catalyze the cleavage of RNA molecules with selected sequences.
  • ribozymes RNA enzymes
  • the cleavage of selected messenger RNAs can result in the reduced production of their encoded polypeptide products.
  • These genes may be used to prepare novel transgenic plants which possess them.
  • the transgenic plants may possess reduced levels of polypeptides including, but not limited to, the polypeptides cited above.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base- pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U. S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • RNA cleavage activity examples include sequences from the Group I self splicing introns including Tobacco RINgspot Virus (Prody et al, 1986), Avocado Sunblotch Viroid (Palukaitis et al, 1979), and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequences from these and related viruses are referred to as hammerhead ribozyme based on a predicted folded secondary structure.
  • ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al, 1992, Yuan and Altman, 1994, U. S. Patents 5,168,053 and 5,624,824), hairpin ribozyme structures (Berzal-Herranz et al, 1992; Chowrira et al, 1994) and Hepatitis Delta virus based ribozymes (U. S. Patent 5,625,047).
  • the general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowrira et al, 1994; Thompson et al , 1995).
  • Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence which is the cleavage site.
  • the cleavage site is a dinucleotide sequence on the target RNA is a uracil (U) followed by either an adenine, cytosine or uracil (A,C or U) (Perriman et al, 1992; Thompson et al, 1995).
  • U uracil
  • A,C or U adenine, cytosine or uracil
  • the frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16. Therefore, for a given target messenger RNA of 1000 bases, 187 dinucleotide cleavage sites are statistically possible.
  • Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al, (1994) and Lieber and Strauss (1995), each incorporated by reference. The identification of operative and preferred sequences for use in down regulating a given gene is simply a matter of preparing and testing a given sequence, and is a routinely practiced "screening" method known to those of skill in the art.
  • genes may be introduced to produce novel transgenic plants which have reduced expression of a native gene product by the mechanism of co-suppression, thus this technique could be used in accordance with the invention. It has been demonstrated in tobacco, tomato, and petunia (Goring et al, 1991; Smith et al, 1990; Napoli et al, 1990; van der Krol et al, 1990) that expression of the sense transcript of a native gene will reduce or eliminate expression of the native gene in a manner similar to that observed for antisense genes.
  • the introduced gene may encode all or part of the targeted native protein but its translation may not be required for reduction of levels of that native protein.
  • Antisense treatments are one way of altering fruit quality and/or ethylene response and the characteristics associated therewith in accordance with the invention.
  • constructs comprising the RIN and/or MC genes in antisense orientation may be used to decrease or effectively eliminate the expression of these genes in a plant.
  • antisense technology may be used to "knock-out" the function of a RIN and/or MC or homologous sequences thereof, thereby causing the delay, protraction or inhibition of fruit ripening and/or decreased ethylene responsiveness or sepal development, as well as the effects associated therewith.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see above) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Suitable methods for plant transformation for use with the current invention are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh et al, 1993), by desiccation/inhibition-mediated DNA uptake (Potrykus et al, 1985), by electroporation (U.S. Patent No. 5,384,253, specifically inco ⁇ orated herein by reference in its entirety), by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Patent No. 5,302,523, specifically incorporated herein by reference in its entirety; and U.S. Patent No.
  • Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
  • the use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et ⁇ l, (1985), Rogers et ⁇ l, (1987) and U.S. Patent No. 5,563,055, specifically inco ⁇ orated herein by reference in its entirety.
  • Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is the preferable method for transformation of dicots, including Arabidopsis, tobacco, tomato, and potato. Indeed, while Agrobacterium-mediated transformation has been routinely used with dicotyledonous plants for a number of years, it has only recently become applicable to monocotyledonous plants. Advances in Agrobacterium-mediated transformation techniques have now made the technique applicable to nearly all monocotyledonous plants. For example, Agrobacterium-mediated transformation techniques have now been applied to rice (Hiei et ⁇ l., 1997; U.S. Patent No.
  • Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al, 1985).
  • recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes.
  • the vectors described (Rogers et ⁇ l., 1987) have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present pu ⁇ oses.
  • Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
  • Krzyzek et al. U.S. Patent No. 5,384,253, inco ⁇ orated herein by reference in its entirety
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells.
  • recipient cells are made more susceptible to transformation by mechanical wounding.
  • friable tissues such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly.
  • pectolyases pectolyases
  • mechanically wounding in a controlled manner.
  • some species which have been transformed by electroporation of intact cells include maize (U.S. Patent No.
  • microprojectile bombardment U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO 94/09699; each of which is specifically inco ⁇ orated herein by reference in its entirety.
  • particles may be coated with nucleic acids and delivered into cells by a propelling force.
  • Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA.
  • DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
  • cells in suspension are concentrated on filters or solid culture medium.
  • immature embryos or other target cells may be arranged on solid culture medium.
  • the cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.
  • An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension.
  • the screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
  • Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species. Examples of species for which have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al, 1985; Lorz et al, 1985; Omirulleh et al, 1993; Fromm et al, 1986; Uchimiya et al, 1986; Callis et al, 1987; Marcotte et al, 1988).
  • Examples of the use of direct uptake transformation of cereal protoplasts include transformation of rice (Ghosh-Biswas et al, 1994), sorghum (Battraw and Hall, 1991), barley (Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh et al, 1993).
  • silicon carbide fiber-mediated transformation may be used with or without protoplasting (Kaeppler, 1990; Kaeppler et al, 1992; U.S. Patent No. 5,563,055, specifically inco ⁇ orated herein by reference in its entirety). Transformation with this technique is accomplished by agitating silicon carbide fibers together with cells in a DNA solution. DNA passively enters as the cell are punctured. This technique has been used successfully with, for example, the monocot cereals maize (PCT Application WO 95/06128, specifically inco ⁇ orated herein by reference in its entirety; Thompson, 1995) and rice
  • site-specific integration techniques could be used to insertionally mutagenize or replace a native RIN or MC gene sequence.
  • An advantage of site- specific integration or excision is that it can be used to overcome problems associated with conventional transformation techniques, in which transformation constructs typically randomly integrate into a host genome in multiple copies. This random insertion of introduced DNA into the genome of host cells can be lethal if the foreign DNA inserts into an essential gene.
  • the expression of a transgene may be influenced by "position effects" caused by the surrounding genomic DNA.
  • Homologous recombination is a reaction between any pair of DNA sequences having a similar sequence of nucleotides, where the two sequences interact (recombine) to form a new recombinant DNA species.
  • the frequency of homologous recombination increases as the length of the shared nucleotide DNA sequences increases, and is higher with linearized plasmid molecules than with circularized plasmid molecules.
  • Homologous recombination can occur between two DNA sequences that are less than identical, but the recombination frequency declines as the divergence between the two sequences increases.
  • Introduced DNA sequences can be targeted via homologous recombination by linking a DNA molecule of interest to sequences sharing homology with endogenous sequences of the host cell.
  • conserved RIN or MC sequences could be used to replace a native RIN or MC sequence with one of the RIN or MC sequences provided herein.
  • the two homologous sequences can interact to insert the introduced DNA at the site where the homologous genomic DNA sequences were located. Therefore, the choice of homologous sequences contained on the introduced DNA will determine the site where the introduced DNA is integrated via homologous recombination.
  • the DNA sequence of interest is linked to DNA sequences sharing homology to a single copy gene of a host plant cell, the DNA sequence of interest will be inserted via homologous recombination at only that single specific site.
  • the DNA sequence of interest is linked to DNA sequences sharing homology to a multicopy gene of the host eukaryotic cell, then the DNA sequence of interest can be inserted via homologous recombination at each of the specific sites where a copy of the gene is located.
  • DNA can be inserted into the host genome by a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • a homologous recombination reaction involving either a single reciprocal recombination (resulting in the insertion of the entire length of the introduced DNA) or through a double reciprocal recombination (resulting in the insertion of only the DNA located between the two recombination events).
  • the introduced DNA should contain sequences homologous to the selected gene.
  • a double recombination event can be achieved by flanking each end of the DNA sequence of interest (the sequence intended to be inserted into the genome) with DNA sequences homologous to the selected gene.
  • a homologous recombination event involving each of the homologous flanking regions will result in the insertion of the foreign DNA.
  • only those DNA sequences located between the two regions sharing genomic homology become integrated into the genome.
  • introduced sequences can be targeted for insertion into a specific genomic site via homologous recombination, in higher eukaryotes homologous recombination is a relatively rare event compared to random insertion events.
  • any transformed cell that contains an introduced DNA sequence integrated via homologous recombination will also likely contain numerous copies of randomly integrated introduced DNA sequences. Therefore, to maintain control over the copy number and the location of the inserted DNA, these randomly inserted DNA sequences can be removed.
  • One manner of removing these random insertions is to utilize a site-specific recombinase system.
  • a site specific recombinase system consists of three elements: two pairs of DNA sequence (the site - specific recombination sequences) and a specific enzyme (the site-specific recombinase). The site-specific recombinase will catalyze a recombination reaction only between two site -specific recombination sequences.
  • a number of different site specific recombinase systems could be employed in accordance with the instant invention, including, but not limited to, the Cre/lox system of bacteriophage PI (U.S. Patent No. 5,658,772, specifically inco ⁇ orated herein by reference in its entirety), the FLP/FRT system of yeast (Golic and Lindquist, 1989), the Gin recombinase of phage Mu (Maeser et al., 1991), the Pin recombinase of E. coli ( ⁇ nomoto et al, 1983), and the R/RS system of the pSRl plasmid (Araki et al, 1992).
  • the bacteriophage PI Cre/lox and the yeast FLP/FRT systems constitute two particularly useful systems for site specific integration or excision of transgenes.
  • a recombinase (Cre or FLP) will interact specifically with its respective site -specific recombination sequence (lox or FRT, respectively) to invert or excise the intervening sequences.
  • the sequence for each of these two systems is relatively short (34 bp for lox and 47 bp for FRT) and therefore, convenient for use with transformation vectors.
  • Experiments on the performance of the FLP/FRT system in both maize and rice protoplasts indicate that FRT site structure, and amount of the FLP protein present, affects excision activity.
  • the systems can catalyze both intra- and intermolecular reactions in maize protoplasts, indicating its utility for DNA excision as well as integration reactions.
  • the recombination reaction is reversible and this reversibility can compromise the efficiency of the reaction in each direction. Altering the structure of the site - specific recombination sequences is one approach to remedying this situation.
  • the site -specific recombination sequence can be mutated in a manner that the product of the recombination reaction is no longer recognized as a substrate for the reverse reaction, thereby stabilizing the integration or excision event.
  • Cre-lox In the Cre-lox system, discovered in bacteriophage PI, recombination between loxP sites occurs in the presence of the Cre recombinase (see, e.g., U.S. Patent No. 5,658,772, specifically inco ⁇ orated herein by reference in its entirety). This system has been utilized to excise a gene located between two lox sites which had been introduced into a yeast genome (Sauer, 1987). Cre was expressed from an inducible yeast GAL1 promoter and this Cre gene was located on an autonomously replicating yeast vector.
  • lox sites on the same DNA molecule can have the same or opposite orientation with respect to each other. Recombination between lox sites in the same orientation results in a deletion of the DNA segment located between the two lox sites and a connection between the resulting ends of the original DNA molecule.
  • the deleted DNA segment forms a circular molecule of DNA.
  • the original DNA molecule and the resulting circular molecule each contain a single lox site. Recombination between lox sites in opposite orientations on the same DNA molecule results in an inversion of the nucleotide sequence of the DNA segment located between the two lox sites.
  • reciprocal exchange of DNA segments proximate to lox sites located on two different DNA molecules can occur. All of these recombination events are catalyzed by the product of the Cre coding region.
  • Modification and changes may be made in the nucleic acids provided by the present invention and accordingly the structure of the polypeptides encoded thereby, and still obtain functional molecules that encode RIN and/or MC polypeptides.
  • the following is a discussion based upon alerting nucleic acids in the RIN and/or MC sequences to result in a changing of the amino acids of a RIN and/or MC polypeptide to create an equivalent, or even an improved, second-generation molecule.
  • mutated RIN or MC proteins are contemplated to be useful for increasing the activity of the protein.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 1.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or f activity. In making such changes, the hydropathic index of amino acids may be considered.
  • hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte et al, 1982, inco ⁇ orate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte et al., 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (—4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein.
  • substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1) glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • peptide mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, "Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • the next steps generally concern identifying the transformed cells for further culturing and plant regeneration.
  • identifying the transformed cells for further culturing and plant regeneration.
  • one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the invention.
  • DNA is introduced into only a small percentage of target cells in any one experiment.
  • a means for selecting those cells that are stably transformed is to introduce into the host cell, a marker gene which confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide.
  • antibiotics which may be used include the aminoglycoside antibiotics neomycin, kanamycin and paromomycin, or the antibiotic hygromycin.
  • aminoglycoside antibiotics Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.
  • NPT II neomycin phosphotransferase II
  • hygromycin phosphotransferase Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphostransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.
  • Potentially transformed cells then are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further
  • Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus and is composed of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. Upon removal of the L-alanine residues by intracellular peptidases, the PPT is released and is a potent inhibitor of glutamine synthetase (GS), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al, 1973). Synthetic PPT, the active ingredient in the herbicide LibertyTM also is effective as a selection agent. Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.
  • the organism producing bialaphos and other species of the genus Streptomyces also synthesizes an enzyme phosphinothricin acetyl transferase (PAT) which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes.
  • PAT phosphinothricin acetyl transferase
  • the use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642 829 A, wherein the gene is isolated from Streptomyces viridochromogenes.
  • this enzyme acetylates the free amino group of PPT preventing auto-toxicity (Thompson et al, 1987).
  • the bar gene has been cloned (Murakami et al, 1986; Thompson et al, 1987) and expressed in transgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (De Block et al, 1989) and maize (U.S. Patent No. 5,550,318).
  • some transgenic plants which expressed the resistance gene were completely resistant to commercial formulations of PPT and bialaphos in greenhouses.
  • Another example of a herbicide which is useful for selection of transformed cell lines in the practice of the invention is the broad spectrum herbicide glyphosate.
  • Glyphosate inhibits the action of the enzyme EPSPS which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived thereof.
  • U.S. Patent No. 4,535,060 describes the isolation of EPSPS mutations which confer glyphosate resistance on the Salmonella typhimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zea mays and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro.
  • Mutant genes encoding glyphosate resistant EPSPS enzymes are described in, for example, International Patent WO 97/4103.
  • the best characterized mutant EPSPS gene conferring glyphosate resistance comprises amino acid changes at residues 102 and 106, although it is anticipated that other mutations will also be useful (PCT/WO97/4103).
  • bombarded tissue is cultured for 0 - 28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg/1 bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1- 3 mg/1 bialaphos or 1-3 mM glyphosate will typically be preferred, it is proposed that ranges of 0.1-50 mg/1 bialaphos or 0.1-50 mM glyphosate will find utility in the practice of the invention.
  • Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium.
  • Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
  • the herbicide DALAPON 2,2-dichloropropionic acid
  • the enzyme 2,2-dichloropropionic acid dehalogenase inactivates the herbicidal activity of 2,2-dichloropropionic acid and therefore confers herbicidal resistance on cells or plants expressing a gene encoding the dehalogenase enzyme (Buchanan- Wollaston et al, 1992; U.S. Patent Appl. No. 08/113,561, filed August 25, 1993; U.S. Patent No. 5,508,468; and U.S. Patent No. 5,508,468; each of the disclosures of which is specifically inco ⁇ orated herein by reference in its entirety).
  • anthranilate synthase which confers resistance to certain amino acid analogs, e.g., 5-methyltryptophan or 6-methyl anthranilate, may be useful as a selectable marker gene.
  • an anthranilate synthase gene as a selectable marker was described in U.S. Patent No. 5,508,468; and U.S. Patent Application 08/604,789.
  • a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media.
  • the R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the C 1 and B genes will result in pigmented cells and/or tissues.
  • the enzyme luciferase may be used as a screenable marker in the context of the present invention.
  • cells expressing luciferase emit light which can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification.
  • the photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.
  • Another screenable marker which may be used in a similar fashion is the gene coding for green fluorescent protein.
  • a selection agent such as bialaphos or glyphosate
  • selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone.
  • combinations of selection and screening may enable one to identify transformants in a wider variety of cell and tissue types. This may be efficiently achieved using a gene fusion between a selectable marker gene and a screenable marker gene, for example, between an NPTII gene and a GFP gene.
  • Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in media that supports regeneration of plants.
  • MS and N6 media may be modified by including further substances such as growth regulators.
  • a preferred growth regulator for such pu ⁇ oses is dicamba or 2,4-D.
  • other growth regulators may be employed, including NAA, NAA + 2,4-D or perhaps even picloram. Media improvement in these and like ways has been found to facilitate the growth of cells at specific developmental stages.
  • Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the mo ⁇ hology of the tissue is suitable for regeneration, at least 2 wk, then transferred to media conducive to maturation of embryoids. Cultures are transferred every 2 wk on this medium. Shoot development will signal the time to transfer to medium lacking growth regulators. The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, will then be allowed to mature into plants.
  • Developing plantlets are transferred to soiless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO , and 25-
  • Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Cons. Regenerating plants are preferably grown at about 19 to 28°C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
  • seeds on transformed plants may occasionally require embryo rescue due to cessation of seed development and premature senescence of plants.
  • To rescue developing embryos they are excised from surface-disinfected seeds 10-20 days post- pollination and cultured.
  • An embodiment of media used for culture at this stage comprises MS salts, 2% sucrose, and 5.5 g/1 agarose.
  • embryo rescue large embryos (defined as greater than 3 mm in length) are germinated directly on an appropriate media. Embryos smaller than that may be cultured for 1 wk on media containing the above ingredients along with 10 "5 M abscisic acid and then transferred to growth regulator-free medium for germination.
  • Progeny may be recovered from transformed plants and tested for expression of the exogenous expressible gene by localized application of an appropriate substrate to plant parts such as leaves.
  • an appropriate substrate such as leaves.
  • transformed parental plants (Ro) and their progeny of any generation tested exhibited no bialaphos-related necrosis after localized application of the herbicide Basta to leaves, if there was functional PAT activity in the plants as assessed by an in vitro enzymatic assay. All PAT positive progeny tested contained bar, confirming that the presence of the enzyme and the resistance to bialaphos were associated with the transmission through the germline of the marker gene.
  • assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCRTM; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
  • Genomic DNA may be isolated from callus cell lines or any plant parts to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note, that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell.
  • PCRTM polymerase chain reaction
  • Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome.
  • the technique of Southern hybridization provides information that is obtained using PCRTM, e.g., the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.
  • RNA will only be expressed in particular cells or tissue types and hence it will be necessary to prepare RNA for analysis from these tissues.
  • PCRTM techniques also may be used for detection and quantitation of RNA produced from introduced genes. In this application of PCRTM it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCRTM techniques amplify the DNA. In most instances PCRTM techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of an RNA species.
  • Southern blotting and PCRTM may be used to detect the gene(s) in question, they do not provide information as to whether the corresponding protein is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.
  • Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins.
  • Unique physical- chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography.
  • the unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification.
  • Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and l4 C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.
  • Bioassays Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the chemical composition, mo ⁇ hology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins which change amino acid composition and may be detected by amino acid analysis, or by enzymes which change starch quantity which may be analyzed by near infrared reflectance spectrometry. Mo ⁇ hological changes may include greater stature or thicker stalks. Most often changes in response of plants or plant parts to imposed treatments are evaluated under carefully controlled conditions termed bioassays.
  • Assays may be employed with the instant invention for determination of the relative efficiency of transgene expression. Such methods would also be useful in evaluating, for example, random or site-specific mutants of the RIN and/or MC sequences provided herein. Alternatively, assays could be used to determine the efficacy of expression when various different enhancers, terminators or other types of elements potentially used in the preparation of transformation constructs.
  • expression assays may comprise a system utilizing embryogenic or non- embryogenic cells, or alternatively, whole plants.
  • An advantage of using cellular assays is that regeneration of large numbers of plants is not required. However, the systems are limited in that promoter activity in the non-regenerated cells may not directly correlate with expression in a plant. Additionally, assays of tissue or developmental specific promoters are generally not feasible.
  • the biological sample to be assayed may comprise nucleic acids isolated from the cells of any plant material according to standard methodologies (Sambrook et al, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic acid is amplified.
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g. , ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • the non- transformed control plants will be of a genetic background similar to the transformed plants. In this way, it is possible to detect differences in the amount or kind of protein detected in various transformed plants.
  • clonal cultures of cells for example, callus or an immature embryo, may be compared to other cells samples.
  • a variety of different assays are contemplated in the screening of cells or plants of the current invention and associated promoters. These techniques may in cases be used to detect for both the presence and expression of the particular genes as well as rearrangements that may have occurred in the gene construct.
  • the techniques include but are not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, pulsed field gel electrophoresis (PFGE) analysis, Southern or Northern blotting, single-stranded conformation analysis (SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.
  • RT Reverse transcription
  • RT-PCRTM relative quantitative PCRTM
  • PCRTM the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting.
  • the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCRTM amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCRTM reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCRTM products and the relative mRNA abundances is only true in the linear range of the PCRTM reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCRTM for a collection of RNA populations is that the concentrations of the amplified PCRTM products must be sampled when the PCRTM reactions are in the linear portion of their curves.
  • the second condition that must be met for an RT-PCRTM study to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • RT-PCRTM The goal of an RT-PCRTM study is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • Most protocols for competitive PCRTM utilize internal PCRTM standards that are approximately as abundant as the target. These strategies are effective if the products of the PCRTM amplifications are sampled during their linear phases. If the products are sampled when the reactions are approaching the plateau phase, then the less abundant product becomes relatively over represented. Comparisons of relative abundances made for many different RNA samples, such as is the case when examining RNA samples for differential expression, become distorted in such a way as to make differences in relative abundances of RNAs appear less than they actually are. This is not a significant problem if the internal standard is much more abundant than the target. If the internal standard is more abundant than the target, then direct linear comparisons can be made between RNA samples.
  • RT-PCRTM assay for plant tissue.
  • the problems inherent in plant tissue samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the RT-PCRTM is performed as a relative quantitative RT-PCRTM with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • RT-PCRTM assays can be superior to those derived from the relative quantitative RT-PCRTM assay with an internal standard.
  • Markers represent an efficient means for assaying the expression of transgenes.
  • a selectable marker one could quantitatively determine the resistance conferred upon a plant or plant cell by a construct comprising the selectable marker coding region operably linked to the promoter to be assayed, e.g., an RS324 promoter.
  • various plant parts could be exposed to a selective agent and the relative resistance provided in these parts quantified, thereby providing an estimate of the tissue specific expression of the promoter.
  • Screenable markers constitute another efficient means for quantifying the expression of a given transgene. Potentially any screenable marker could be expressed and the marker gene product quantified, thereby providing an estimate of the efficiency with which the promoter directs expression of the transgene. Quantification can readily be carried out using either visual means, or, for example, a photon counting device.
  • a preferred screenable marker gene assay for use with the current invention constitutes the use of the screenable marker gene ⁇ -glucuronidase (GUS). Detection of GUS activity can be performed histochemically using 5-bromo-4-chloro-3-indolyl glucuronide (X- gluc) as the substrate for the GUS enzyme, yielding a blue precipitate inside of cells containing GUS activity. This assay has been described in detail (Jefferson, 1987). The blue coloration can then be visually scored, and estimates of expression efficiency thereby provided. GUS activity also can be determined by immunoblot analysis or a fiuorometric GUS specific activity assay (Jefferson, 1987).
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • Gel chromatography is unsu ⁇ assed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adso ⁇ tion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequences set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
  • Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 under relatively stringent conditions such as those described herein.
  • sequences may encode the entire RIN and/or MC protein or functional or non-functional fragments thereof.
  • the hybridizing segments may be shorter oligonucleotides. Sequences of 17 bases long should occur only once in the genome of most plant species and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length.
  • exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, or 3000 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
  • Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl , at temperatures ranging from approximately 40°C to about 72°C.
  • Formamide and SDS also may be used to alter the hybridization conditions.
  • One method of using probes and primers of the present invention is in the search for genes related to RIN and/or MC from other species.
  • the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules.
  • screening may involve analysis of RNA molecules.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site- specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • transgenic plants may be made by crossing a plant having a construct of the invention to a second plant lacking the construct.
  • a nucleic acid sequence encoding a RIN and/or MC coding sequence can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the current invention not only encompasses a plant directly created from cells which have been transformed in accordance with the current invention, but also the progeny of such plants.
  • progeny denotes the offspring of any generation of a parent plant prepared in accordance with the instant invention, wherein the progeny comprises a construct prepared in accordance with the invention.
  • Crossing a plant to provide a plant line having one or more added transgenes relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a transgene of the invention being introduced into a plant line by crossing a starting line with a donor plant line that comprises a transgene of the invention. To achieve this one could, for example, perform the following steps:
  • backcrossing is herein defined as the process including the steps of: (a) crossing a plant of a first genotype containing a desired gene, DNA sequence or element to a plant of a second genotype lacking said desired gene, DNA sequence or element;
  • step (d) repeating steps (b) and (c) for the pu ⁇ ose of transferring said desired gene, DNA sequence or element from a plant of a first genotype to a plant of a second genotype.
  • Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion.
  • a plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid.
  • a plant genotype lacking said desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.
  • a vector or expression cassette into a cell or protoplast in which that exogenous DNA is inco ⁇ orated into a chromosome or is capable of autonomous replication.
  • Exogenous gene A gene which is not normally present in a given host genome in the exogenous gene's present form In this respect, the gene itself may be native to the host genome, however, the exogenous gene will comprise the native gene altered by the addition or deletion of one or more different regulatory elements.
  • Expression The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.
  • Expression vector A nucleic acid comprising one or more coding sequences which one desires to have expressed in a transgenic organism.
  • Progeny Any subsequent generation, including the seeds and plants therefrom, which is derived from a particular parental plant or set of parental plants.
  • Promoter A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
  • Ro Transgenic Plant A plant which has been directly transformed with a selected
  • DNA or has been regenerated from a cell or cell cluster which has been transformed with a selected DNA has been regenerated from a cell or cell cluster which has been transformed with a selected DNA.
  • Regeneration The process of growing a plant from a plant cell (e.g., plant protoplast, callus or explant).
  • Selected DNA A DNA which one desires to have expressed in a transgenic plant, plant cell or plant part.
  • a selected DNA may be native or foreign to a host genome, but where ' the selected DNA is present in the host genome, may include one or more regulatory or functional elements which alter the expression profile of the selected gene relative to native copies of the gene.
  • Selected Gene A gene which one desires to have expressed in a transgenic plant, plant cell or plant part.
  • a selected gene may be native or foreign to a host genome, but where the selected gene is present in the host genome, will include one or more regulatory or functional elements which differ from native copies of the gene.
  • Transformation construct A chimeric DNA molecule which is designed for introduction into a host genome by genetic transformation.
  • Preferred transformation constructs will comprise all of the genetic elements necessary to direct the expression of one or more exogenous genes, for example, RIN and/or MC genes. Included within in this term are, for example, expression cassettes isolated from a starting vector molecule.
  • Transformed cell A cell the DNA complement of which has been altered by the introduction of an exogenous DNA molecule into that cell.
  • Transgene A segment of DNA which has been inco ⁇ orated into a host genome or is capable of autonomous replication in a host cell and is capable of causing the expression of one or more cellular products. Exemplary transgenes will provide the host cell, or plants regenerated therefrom, with a novel phenotype relative to the corresponding non-transformed cell or plant. Transgenes may be directly introduced into a plant by genetic transformation, or may be inherited from a plant of any previous generation which was transformed with the DNA segment.
  • Transgenic plant A plant or progeny plant of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof contains an introduced exogenous DNA segment not originally present in a non-transgenic plant of the same strain.
  • the transgenic plant may additionally contain sequences which are native to the plant being transformed, but wherein the "exogenous" gene has been altered in order to alter the level or pattern of expression of the gene.
  • Transit peptide A polypeptide sequence which is capable of directing a polypeptide to a particular organelle or other location within a cell.
  • C represents a DNA pool derived from 5 individuals with ripening fruit
  • e and c designate the L. esculentum (mutant) and L. cheesmannii (normal) alleles of the RFLP probes employed, respectively.
  • Hybridization to the "c" allele of TG448 in pools 1, 3 and 6 represents individuals within these pools which have undergone recombination between the TG448 and rin loci. The entire set of 45 pools was used to generate an RFLP map of the rin region of chromosome 5.
  • DNA pools nearly isogenic for all three ripening locus regions were generated and screened for polymo ⁇ hic RAPD products.
  • Random primers will be purchased from Operon Technologies and the population used for the generation of isogenic DNA pools is the L. esculentum X L. pennellli F2 mapping population used to generate the tomato RFLP map. This population will be used rather than the actual ripening gene mapping populations because of the higher degree of polymo ⁇ hism between the parents (Miller and Tanksley, 1990). The pu ⁇ ose of this effort will be to identify tightly linked molecular probes to be utilized in chromosome walks. In previous analysis employing tomato genomic DNA templates, 3-7 amplification products were detected, on average, per primer.
  • 500 primers should result in approximately 2,500 amplified loci of the tomato genome. Given the estimated map size of 1500 cM for the tomato genome, 500 primers should yield approximately 1-2 markers within 1 cM on either side of a target locus. In the event that sufficient polymo ⁇ hic markers are not identified for one or more of the target loci, additional primers will be acquired through mutual primer exchanges. Also, the use of random primer pairs has recently been demonstrated to yield significant numbers of unique amplification products from tomato genomic DNA.
  • a total of 100 random 10 base primers were utilized for amplification of nearly isogenic DNA pools resulting in one polymo ⁇ hic PCR product, P76.
  • the 0.5 kb P76 amplification product was gel-purified, labeled and mapped via an EcoRI RFLP to the target region.
  • a 360 kb clone (Yrin2) demonstrated hybridization to both TG503 and CT63 suggesting that 1) it contained sequences closer to the target locus than any of the other clones, and 2) the estimated 191 kb/cM ratio in the region of RIN was within two fold of accurate (i.e. TG503 and CT63 are separated by 1 cM and reside on a 360 kb YAC).
  • Yrw2R A single copy end of Yrw2 was isolated by inverse PCR and mapped, in a population of 670 F2 progeny, on the RIN side of CT63 0.2 cM from rin.
  • cDNA libraries in the vector lambda gtlO and made from Mature Green, Breaker, and Red Ripe stage fruit are all available in the laboratory (lambda gtlO does not cross hybridize with the YAC vector pYAC4). All 3 libraries will be screened because it is not known which stage will express the highest levels of target gene product. Construction of a fourth cDNA library may also be used in lambda gtlO made from mRNA derived from several stages of immature fruit development. Specifically, mRNA will be combined from ovaries prior to pollination, 2 days post pollination, and every 10 days post-pollination up to the Mature Green stage (approximately 30 days in cultivar Ailsa Craig). This library will help to minimize the problem of not knowing when during fruit development the RIN or NOR gene is expressed. Tissue for all but the unpollinated ovaries are stored in a -80°C freezer.
  • CT16 Prior to initiation of the chromosome walk to nor, the closest known flanking markers were CT16 (0.9 cM) and CT41 (2.3 cM). Based on the published tomato RFLP map (Tanksley et al, 1992), it was known that TG395 resided in the interval between CT41 and CT16 and thus represented a closer marker to NOR than at least one of the two. TG395 was mapped as a sequence tagged site (due to lack of RFLPs between the two parents of the mapping population) to 1.4 cM from NOR on the CT41 side. PCR screening of the tomato YAC library with TG395 specific primers resulted in the identification of 7 YAC clones ranging in size from 50 kb - 490 kb. CT16 was not used for library screening because of difficulty in generating reliable PCR primers.
  • YAC end and RFLP marker cross hybridizations yielded a YAC contig.
  • TG395 which was used to retrieve this clone from the library
  • CT16 CT16
  • a high titer cosmid library (>500,000 clones) was then prepared of the yeast containing Ynor3 to use in fine mapping and walking to NOR within the Ynor3 clone.
  • The, cosmid vector employed, 04541 contains T-DNA borders to permit direct Agrobacterium transfer into plants. Random Ynor3 Pstl and Haelll subclones were also isolated to use as fine mapping probes.
  • a "breaker" stage tomato fruit cDNA library was also screened with Ynor3 as probe, yielding numerous clones for characterization for linkage to nor.
  • Yrinl 1 a YAC termed Yrinl 1 was identified which harbored the rin locus as determined by random subcloning of Yrinl 1 restriction fragments and RFLP mapping to determine that sequences flanking the target locus were contained on this YAC.
  • This YAC also contained sequences that were absent from the YAC descried above with an internal deletion (thus confirming the integrity of Yrinl 1), and said fragments were tightly linked to the rin locus.
  • cDNAs hybridizing to the two candidate gene YACs, and thus potentially representing target gene transcripts, were verified for homology via hybridization as probes back to the respective Yrinl 1 and Ynor3 YACs. Positives were mapped as RFLPs and sequenced.
  • cDNA clones hybridizing to Yrinl 1 were identified via hybridization to a tomato breaker fruit cDNA library as described above for Ynor3. Positives were sequenced and several homologous to AGL (agamous-like) MADS box genes were identified. Two separate classes of tomato AGLs were identified from this group and are represented by cDNAs rin34 (SEQ ID NO:7) and rin43 (the original sequences obtained are given by SEQ ID NO:3 and the corrected sequences are given in SEQ ID NO:5 and SEQ ID NO:6). DNA gel-blot hybridization with subclones of both the rin43 and rin34 genes indicates that parts of both genes are deleted from the rin genome.
  • RNA gel-blot analysis indicated that an alternate transcript size (higher MW) could be identified in the rin/rin line as compared to normal (RIN/RIN) using either probe.
  • both rin43 and rin34 are induced during ripening and by ethylene in mature green fruit.
  • RT-PCR using a primer from the 5' end of rin43 and the 3' end of rin34 yielded a product only from the rin/rin line. The resulting product represents an in-frame fusion of the 5' end of rin43 and the 3' end of rin34 sequences.
  • results were inte ⁇ reted to mean that a deletion has resulted in loss of the 3' portion of rin43 and the 5' portion of rin34 resulting in the creation of an in-frame chimeric 43/34 gene under direction of the rin43 promoter.
  • the rin fruit ripening phenotype is due to the lesion in the rin43 sequence
  • the inventors also believe that the lack of ripening in rin may be due to mutation in 1) rin43, 2) rin34, 3) both rin43 and rin34, and 4) it is possible that the phenotype results from expression of the rin43/34 chimeric transcript.
  • EcoRV and Sad sites of the pBluescript vector containing the RIN cD ⁇ A were employed due to the fact that Smal and EcoRV (blunt) ends are compatible for ligation.
  • the resulting ligated sequence no longer can be digested with Smal or EcoRV.
  • Completed sense and antisense constraints were initially transformed into E. coli DH 10B cells and then resulting plasmid D ⁇ A was isolated and transformed into Agrobacterium tumefaciens strain LBA 4404 for use in transfer into the tomato genome as described herein.
  • Sad sites of PB1121 (following removal of the GUS gene from pBI121).
  • 3 bp of pBluescript polylinker (BS) was included on the Smal side of the cD ⁇ A and 51 bp of pBluescript polylinker was included on the Sad side of the insert (including the following restriction sites: SacII, ⁇ otI, Xbal, Spel, BamHI, Saml, Pstl, EcoRI).
  • MC-containing sense and antisense constructs were made by replacing the GUS gene of T-DNA binary vector pBI121 with the full-length MC cDNA, referred to as CD-34 (1027 bp) in sense and antisense orientations, respectively, relative to the CaMV35S promoter (35S- P) of pBI121 (FIG. 2).
  • CD-34 1027 bp
  • the MC cDNA was subcloned between Smal and Sad restriction sites resulting from removal of the GUS gene from pBI121.
  • Completed sense and antisense constructs were initially transformed into E. coli DH 10B cells and then resulting plasmid DNA was isolated and transformed into Agrobacterium tumefaciens strain LBA 4404 for use in transfer into the tomato genome as described herein.
  • the sense and antisense orientations of the MC cDNA sequence relative to ⁇ coRV and Smal restriction sites were obtained by subcloning the original MC cDNA bound by the ⁇ coRI sites from the original cDNA library vector (lambda gtlO) into the ⁇ coRI site of plasmid vector pBluescript. Due to the fact that the cDNA sequence was flanked by identical restriction sites ( ⁇ coRI), the insert could insert in either direction essentially at random.
  • MC-pBluescript clones were isolated and sequenced to determine the orientation of the cDNA insert relative to the ⁇ coRV and Sad sites of pBluescript, and one clone in which the 5' end of the cDNA was adjacent to the Smal site of pBluescript was selected.
  • the GUS gene of pBI121 was premoved via Sad and Smal digestion and the resulting plasmid was treated with single-stranded DNA exonuclease to remove the 3' overhang of Sad (Sad (M) resulting in blunt end DNA at both the Smal and Sad ends of pBI121).
  • the MC cDNA was released from pBluescript with Smal and ⁇ coRV (thus also blunt end on both sides) and ligated into the blunt end pBI121 plasmid. Resulting clones were sequenced to identify clones which contained the MC cDNA in either the sense or antisense orientation and one of each orientation was used for tomato transformation.
  • the MC cDNA contains an internal Sad site so this enzyme could not be used in construct development.
  • a number of RIN and MC transformation vectors were prepared by the inventors for studies of the function of these genes. Prepared vectors included those given in Table 2, below.
  • Transgenic tomato plant were prepared as follows. First, the explant was prepared by sterilizing seeds with soaking in 20% bleach + 0.1 % Tween-20 for 15 minutes. The seeds were rinses 4 times in sterile distilled H20 and the seeds sown on MSO medium ( (1 Liter): 4.0 g MS Salts (Gibco), 5.0 ml B5 Vitamins, 5.0 ml MS Iron/EDTA, 20.0 g sucrose, 7.0 g agar (phytagar), pH medium to 6.0 with KOH, autoclave) in sterile glass jars, grow in growth chamber. Agrobacterium was then prepared by streaking selective fresh plates with Agrobacterium containing the desired construct for 2-3 days before it was needed.
  • MSO medium (1 Liter): 4.0 g MS Salts (Gibco), 5.0 ml B5 Vitamins, 5.0 ml MS Iron/EDTA, 20.0 g sucrose, 7.0 g agar (phytagar), pH medium to 6.0 with KOH, autoclave
  • a single colony was picked from the plate and grown in tubes containing 2 ml YEP medium (YEP Rich Medium (500 ml): 5.0 g Bacto-peptone, 2.5 g NaCl, 5.0 g Bacto Yeast Extract, 7.5 g Bacto agar, autoclave) with appropriate antibiotics, followed by incubation on a shaker at 28°C overnight. Explants were precultured two days before infection takes place, and 8-10 day old cotyledons were excised. With sterile forceps, cotyledons were removed and placed onto an MSO plate. Using forceps and a blade, the cotyledons were cut in 1-2 pieces.
  • YEP medium YEP Rich Medium (500 ml): 5.0 g Bacto-peptone, 2.5 g NaCl, 5.0 g Bacto Yeast Extract, 7.5 g Bacto agar, autoclave
  • Explants were precultured two days before infection takes place, and 8
  • cotyledon pieces were placed on co-cultivation media ((500 ml): 500 ml MSO Medium, 1 ml KH2PO4 (100 mg/ml), 250 (1 Kinetin (0.2 mg/ml), 100 (1 2,4 D (1 mg/ml), 735 (1 acetosyringone)), wrapped in parafilm and placed in growth chamber for 2-3 days.
  • co-cultivation media ((500 ml): 500 ml MSO Medium, 1 ml KH2PO4 (100 mg/ml), 250 (1 Kinetin (0.2 mg/ml), 100 (1 2,4 D (1 mg/ml), 735 (1 acetosyringone)
  • Regeneration Medium (1Z) 500 ml MSO Medium, 5.0 ml Carbenicillin stock, 1.0 ml Kanamycin stock, 0.5 ml Zeatin (1 mg/ml), 0.1 ml IAA
  • Regeneration Medium (1Z) 500 ml MSO Medium, 5.0 ml Carbenicillin stock, 1.0 ml Kanamycin stock, 0.5 ml Zeatin (1 mg/ml), 0.1 ml IAA
  • Plantlets were covered with magenta box to conserve higher humidity. After 5-7 days, the magenta box was gradually removed. Plants were transferred to the greenhouse grown to 10-15 cm. Media used included the following: YEB Rich Medium (500 ml): 2.75g Beef Extract, 0.55g Yeast Extract, 2.75g peptone, 2.75g sucrose, 1 ml MgSO4 (1M) pH 7.2, 7.5 g Bactoagar, autoclave. B5 vitamins (100 ml): 2.0 g myo-inositol, 0.2 g thiamine-HCl, 20.0 mg nicotinic acid, 20.0 mg pyridoxine-HCl, mix, then put in autoclaved bottle.
  • IAA Indoleacetic acid
  • IAA Indoleacetic acid
  • Acetosyringone stock (ACE) (lOmg/ml): weigh out 100 mg of acetosyringone, add 10 ml 70% ethanol, filter sterilize, put in 1.5 ml tubes, place in -20C freezer.
  • Carbenicillin Stock (50mg/ml): weigh out 5 grams Carbenicillin, add 100 ml ddH20, filter sterilize, put in 12 ml tubes, place in -20°C freezer.
  • Kanamycin Stock 50mg/ml: weigh out 5 grams Kanamycin, add 100 ml ddH20, filter sterilize, put in 12 ml tubes, place in -20C freezer.
  • Tetracycline Stock (3mg/ml): dissolve 3 mg tetracycline in 1 ml ddH20, filter sterlize, refrigerate in foil (light sensitive), good 1 day.
  • FIG. 3 The results of manipulation of fruit ripening and carotenoid accumulation with the tomato RIN gene are shown in FIG. 3.
  • the top panel shows fruit from five independently transformed tomato lines (A-E) of the genotype rin/rin; mc/cm in the cultivar Ailsa Craig and transformed with i?/N-pBI121 Sense (FIG. 1).
  • Primary transformants (TO) were confirmed for transgene integration via D ⁇ A gel-blot analysis and subsequently self-pollinated. Resulting seed were harvested and grown (TI generation) and analyzed for transgene segregation. Representative fully mature fruit from TI rin/rin; mc/mc individuals that harbor the sense i?/N transgene (top) or have segregated it out (bottom) are shown.
  • the results demonstrate that transgene expression in the mutant background recovers the non- ripening phenotype and confers ripening. Integration of the same transgene in multiple independent lines has a range of effects shown in A-E.
  • the bottom panel shows representative mature fruit from normal (Rin/Rin) and nearly isogenic mutant (rin/rin) Ailsa Craig tomato lines.
  • the fruit marked SS is a representative fruit from a normal (Rin/Rin; Mc/Mc) line also transformed with the i?/N-pBI121 Sense construct but which shows inhibited ripening.
  • R ⁇ A gel-blot analysis indicated that fruit from this line was repressed in RIN gene expression suggestive of a sense-repression (co-suppression) effect. Note that while expression of the normal RIN transgene recovers ripening in mutant fruit, the large sepal phenotype remains (compare the sepals of A-E top to RIN/RIN).
  • FIG. 4 The results of manipulation of sepal development and inflorescence meristem determinacy with the MC gene are indicated in FIG. 4.
  • An antisense MC gene construct (MC- pBI121, FIG. 2) was transferred into the normal tomato cultivar Ailsa Craig. Primary transformants (TO) were confirmed for transgene integration via DNA gel-blot analysis and subsequently self-pollinated. Resulting seed were harvested and grown (TI generation) and analyzed for transgene segregation. Representative floral inflorescences from TI individuals and relevant controls are shown.
  • the left panel shows whole inflorescences (top, middle) and close-ups of sepals (bottom) from two independent TI lines (left and right respectively) which contain (+) or have segregated out (-) the MC-pBI121 antisense gene construct. Note that shoots (leaves) developed from the two (+) inflorescences (middle) but not from those which have segregated out the transgene (-, top).
  • the right panel shows the normal (Rin/Rin) and mutant (rin/rin) controls which have genotypes Rin/Rin; Mc/Mc and rin/rin; mc/mc, respectively.
  • the image labeled (S) is a representative mutant transgenic line (i.e., genotype rin/rin; mc/mc) into which a sense MC transgene has been introduced (MC-pBI121 Sense, FIG. 2) with relatively normal sized sepals and inflorescence determinacy (i.e., mutant mc phenotype recovered), yet which are undergoing premature and dramatic senescence.
  • the expression of the RIN (CD43) and MC (CD34) genes in normal and mutant fruit is indicated in FIG. 6.
  • RNA gel-blot analysis is shown of expression in mature green (MG), mature green plus 8 hours of 20 ppm ethylene (MG+E), breaker or early ripening (BK), and red-ripe (RR) fruit from nearly isogenic normal (WT) and equivalent age fruit homozygous for the nor, rin, and Nr mutations. Note the alteration in transcript size in the rin mutant due to the partial deletion/fusion of RIN and MC sequences as shown in FIG. 7.
  • Genomic DNA sequences corresponding to the i?/N cD ⁇ A were also isolated from the tomato genomic library whose construction is described below. Full length cD ⁇ As were sequenced at their termini, and oligonucleotide primers synthesized corresponding to the 5' and 3' ends. Candidate genomic clones were then utilized as a template in sequencing reactions with these end primers. Those genomic clones harboring D ⁇ A sequences from both ends of the corresponding full length cD ⁇ A, as determined by sequencing, were restriction mapped to identify the location of the transcribed region within the genomic clone insert.
  • a library of tomato genomic DNA was constructed, for use in isolating genomic clones corresponding to cDNAs and in cosmid clone walking (see below), in the cosmid/transformation vector 04541 (derived from the binary Agrobacterium transformation vector pJJ1881; Jones et al, 1992).
  • Genomic DNA (UC82B) was partially digested with Sau3A and size selected to 15-20 kb.
  • the library was subdivided into pools to facilitate efficient screening using PCR or DNA gel-blot hybridization. Approximately 500,000 unamplified clones were plated at a density of 2,000 - 3,000 colonies per plate, resulting in approximately 200 pools. The pools were extracted for DNA to be used in both PCR and DNA gel-blot screening, and maintained as glycerol stocks For immediate piuposes, the primary interest was in the former use, although the latter permits screening at lower stringency and with heterologous probes. In addition, this library, as well as similar ones, will served to complement the other in the event that one is deficient of particular sequences.
  • the walk from CDnor2 was initiated in the Ynor3 yeast cosmid library, while that from Yrin ⁇ L was performed in the tomato genomic cosmid library described above because RIN may have been off the end of Yrw8.
  • a D ⁇ A sequence surrounding the 04541 cloning site was generated as were nested primers for IPCR of insert ends.
  • Backcrossing can be used to improve a starting plant.
  • Backcrossing transfers a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention.
  • the progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent, then the selected progeny are mated back to the superior recurrent parent (A).
  • the progeny are hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes.
  • the last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, /. e. one or more transformation events.
  • transgenes are valuable in that they typically behave genetically as any other gene and can be manipulated by breeding techniques in a manner identical to any other gene. Therefore, one may produce inbred plants which are true breeding for one or more transgenes. By crossing different inbred plants, one may produce a large number of different hybrids with different combinations of transgenes. In this way, plants may be produced which have the desirable agronomic properties frequently associated with hybrids ("hybrid vigor"), as well as the desirable characteristics imparted by one or more transgene(s).
  • Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.
  • Marker assisted breeding may be undertaken as follows. Seed of plants with the desired trait are planted in soil in the greenhouse or in the field. Leaf tissue is harvested from the plant for preparation of DNA at any point in growth at which approximately one gram of leaf tissue can be removed from the plant without compromising the viability of the plant. Genomic DNA is isolated using a procedure modified from Shure et al (1983). Approximately one gram of leaf tissue from a seedling is lypholyzed overnight in 15 ml polypropylene tubes. Freeze-dried tissue is ground to a powder in the tube using a glass rod.
  • Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0 urea, 0.35 M NaCI, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine).
  • Tissue/buffer homogenate is extracted with 3 ml phenol/chloroform.
  • the aqueous phase is separated by centrifugation, and precipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2, and an equal volume of isopropanol.
  • the precipitate is washed with 75% ethanol and resuspended in 100-500 ⁇ l TE (0.01 M Tris- HCl, 0.001 M EDTA, pH 8.0).
  • Genomic DNA is then digested with a 3-fold excess of restriction enzymes, electrophoresed through 0.8% agarose (FMC), and transferred (Southern, 1975) to Nytran (Schleicher and Schuell) using 10X SCP (20 SCP: 2M NaCI, 0.6 M disodiu phosphate, 0.02 M disodium EDTA).
  • 10X SCP 20 SCP: 2M NaCI, 0.6 M disodiu phosphate, 0.02 M disodium EDTA.
  • the filters are prehybridized in 6X SCP, 10% dextran sulfate, 2% sarcosine, and 500 ⁇ g/ml denatured salmon sperm DNA and P-labeled probe generated by random priming (Feinberg & Vogelstein, 1983).
  • Hybridized filters are washed in 2X SCP, 1% SDS at 65° for 30 minutes and visualized by autoradiography using Kodak XAR5 film. Genetic polymo ⁇ hisms which are genetically linked to traits of interest are thereby used to predict the presence or absence of the traits of interest.
  • Southern blot can be stripped of radioactive probe following autoradiography and re-probed with a different probe. In this manner one may identify each of the various transgenes that are present in the plant. Further, one of skill in the art will recognize that any type of genetic marker which is polymo ⁇ hic at the region(s) of interest may be used for the pu ⁇ ose of identifying the relative presence or absence of a trait, and that such information may be used for marker assisted breeding. Each lane of a Southern blot represents DNA isolated from one plant.
  • the integration event composition of each plant may be determined. Correlations may be established between the contributions of particular integration events to the phenotype of the plant. Only those plants that contain a desired combination of integration events may be advanced to maturity and used for pollination. DNA probes corresponding to particular transgene integration events are useful markers during the course of plant breeding to identify and combine particular integration events without having to grow the plants and assay the plants for agronomic performance.
  • restriction enzymes will be used to digest genomic DNA, either singly or in combinations.
  • One of skill in the art will recognize that many different restriction enzymes will be useful and the choice of restriction enzyme will depend on the DNA sequence of the transgene integration event that is used as a probe and the DNA sequences in the genome surrounding the transgene. For a probe, one will want to use DNA or RNA sequences which will hybridize to the DNA used for transformation. One will select a restriction enzyme that produces a DNA fragment following hybridization that is identifiable as the transgene integration event. Thus, particularly useful restriction enzymes will be those which reveal polymo ⁇ hisms that are genetically linked to specific transgenes or traits of interest.
  • EXAMPLE 10 General Methods for Assays DNA analysis of transformed plants is performed as follows. Genomic DNA is isolated using a procedure modified from Shure, et al, 1983. Approximately 1 gm callus or leaf tissue is ground to a fine powder in liquid nitrogen using a mortar and pestle. Powdered tissue is mixed thoroughly with 4 ml extraction buffer (7.0 M urea, 0.35 M NaCI, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, % sarcosine). Tissue/buffer homogenate is extracted with 4 ml phenol/ chloroform.
  • extraction buffer 7.0 M urea, 0.35 M NaCI, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, % sarcosine.
  • the aqueous phase is separated by centrifugation, passed through Miracloth, and precipitated twice using 1/10 volume of 4.4 M ammonium acetate, pH 5.2 and an equal volume of isopropanol.
  • the precipitate is washed with 70% ethanol and resuspended in 200-500 ⁇ l TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0).
  • PCR polymerase chain reaction
  • the reaction is run in a thermal cycling machine as follows: 3 minutes at 94°C, 39 repeats of the cycle 1 minute at 94°C, 1 minute at 50°C, 30 seconds at 72°C, followed by 5 minutes at 72°C. Twenty ⁇ l of each reaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mM Tris-borate, 2 mM EDTA) at 50V for two to four hours.
  • genomic DNA is digested with a 3 -fold excess of restriction enzymes, electrophoresed through 0.8% agarose (FMC), and transferred (Southern, 1975) to Nytran (Schleicher and Schuell) using 10X SCP (20X SCP: 2 M NaCI, 0.6 M disodium phosphate, 0.02 M disodium EDTA).
  • 10X SCP 20X SCP: 2 M NaCI, 0.6 M disodium phosphate, 0.02 M disodium EDTA.
  • Probes are labeled with P using the random priming method (Boehringer Mannheim) and purified using Quik-Sep® spin columns (Isolab Inc., Akron, OH).
  • Filters are prehybridized at 65°C in 6X SCP, 10% dextran sulfate, 2% sarcosine, and 500 ⁇ g/ml heparin (Chomet et al, 1987) for 15 min. Filters then are hybridized overnight at 65 C in 6X SCP containing 100 ⁇ g/ml denatured salmon sperm DNA and 32 P-labeled probe. Filters are washed in 2X SCP, 1% SDS at 65 C for 30 min. and visualized by autoradiography using Kodak XAR5 film. For rehybridization, the filters are boiled for 10 min. in distilled H O to remove the first probe and then prehybridized as described above.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • McCormac et al Euphytica, v. 99 (1) p. 17-25, 1998. McCormick, S., Neidermeyer, J., Fry, J., Barnason, A., Horsch, R. and Frayley, R. (1986)
  • Yamada et al Plant Cell Rep., 4:85, 1986. Yamaguchi-Shinozaki et al, Plant Cell Physiol, 33:217-224, 1992. Yang and Russell, Proc. Nat 'l Acad. Sci. USA, 87:4144-4148, 1990.

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Abstract

L'invention porte: sur des séquences d'acides nucléiques codant pour les gènes RIN et MC; sur des compositions comportant ces séquences; sur des plantes transformées à l'aide desdites séquences; sur des procédés d'expression des gènes RIN et MC incluant la création de plantes transgéniques à l'aide des gènes RIN et MC par transformation génétique ainsi que des procédés de sélection. Les séquences objets de l'invention sont un nouvel outil important de création de plantes transgéniques présentant de préférence une ou plusieurs caractéristiques bénéfiques additionnelles.
PCT/US2000/019035 1999-07-12 2000-07-12 Compositions de genes rin et leurs procedes d'utilisation WO2001004315A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008125433A1 (fr) * 2007-04-16 2008-10-23 Unilever N.V. Tissu de plante présentant une teneur en composant de type flavonoïde modifiée
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
WO2013156204A1 (fr) * 2012-04-17 2013-10-24 Nunhems B.V. Plantes solanum lycopersicum à altérations non-transgéniques dans le gène rin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021794A1 (fr) * 1993-03-22 1994-09-29 Zeneca Limited Adn, produits de recombinaison d'adn, cellules et plantes derivees
WO1999004003A1 (fr) * 1997-07-18 1999-01-28 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021794A1 (fr) * 1993-03-22 1994-09-29 Zeneca Limited Adn, produits de recombinaison d'adn, cellules et plantes derivees
WO1999004003A1 (fr) * 1997-07-18 1999-01-28 Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) Procede d'obtention de plantes transgeniques dont la floraison est inhibee, et sequences d'adn utilisees dans ledit procede

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL SEQUENCE DATABASE [Online] 29 March 2000 (2000-03-29) VAN DER HOEVEN, R.S., ET AL. : "generation of ESTs from tomato flower tissue, 3-8 mm buds - unpublished" XP002157278 *
DATABASE EMBL SEQUENCE DATABASE [Online] 6 January 1999 (1999-01-06) WU, Y.H., ET AL. : "untitled" XP002157276 *
DATABASE EMBL SEUENCE DATABASE [Online] 14 December 1999 (1999-12-14) ALCALA, J., ET AL. : "generation of ESTs from tomao fruit tissue - unpublished" XP002157277 *
GIOVANNONI JAMES J ET AL: "Molecular genetic analysis of the ripening-inhibitor and non-ripening loci of tomato: A first step in genetic map-based cloning of fruit ripening genes." MOLECULAR & GENERAL GENETICS, vol. 248, no. 2, 1995, pages 195-206, XP002157273 ISSN: 0026-8925 cited in the application *
GRAY J E ET AL: "The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening." PLANT CELL AND ENVIRONMENT, vol. 17, no. 5, 1994, pages 557-571, XP000614247 ISSN: 0140-7791 *
HUANG HAI ET AL: "The Arabidopsis MADS-box gene AGL3 is widely expressed and encodes a sequence-specific DNA-binding protein." PLANT MOLECULAR BIOLOGY, vol. 28, no. 3, 1995, pages 549-567, XP002157275 ISSN: 0167-4412 *
TANKSLEY ET AL: "High density molecular linkage maps of the tomato and poato genomes" GENETICS,GENETICS SOCIETY OF AMERICA, AUSTIN, TX,US, vol. 132, December 1992 (1992-12), pages 1141-1160, XP002097980 ISSN: 0016-6731 *
TANKSLEY S D ET AL: "Chromosome landing: a paradigm for map-based gene cloning in plants with large genomes" TRENDS IN GENETICS,NL,ELSEVIER, AMSTERDAM, vol. 11, no. 2, February 1995 (1995-02), pages 63-68, XP004207296 ISSN: 0168-9525 *
THOMPSON ANDREW J ET AL: "Molecular and genetic characterization of a novel pleiotropic tomato-ripening mutant." PLANT PHYSIOLOGY (ROCKVILLE), vol. 120, no. 2, June 1999 (1999-06), pages 383-389, XP002157274 ISSN: 0032-0889 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8022274B2 (en) 1998-09-22 2011-09-20 Mendel Biotechnology, Inc. Plant tolerance to low water, low nitrogen and cold
WO2008125433A1 (fr) * 2007-04-16 2008-10-23 Unilever N.V. Tissu de plante présentant une teneur en composant de type flavonoïde modifiée
WO2013156204A1 (fr) * 2012-04-17 2013-10-24 Nunhems B.V. Plantes solanum lycopersicum à altérations non-transgéniques dans le gène rin
CN104640440A (zh) * 2012-04-17 2015-05-20 纽海姆有限公司 在rin基因中具有非转基因改变的番茄植物

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