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WO1993016096A1 - Adn de gibberelline recombinante et utilisations - Google Patents

Adn de gibberelline recombinante et utilisations Download PDF

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Publication number
WO1993016096A1
WO1993016096A1 PCT/US1993/001121 US9301121W WO9316096A1 WO 1993016096 A1 WO1993016096 A1 WO 1993016096A1 US 9301121 W US9301121 W US 9301121W WO 9316096 A1 WO9316096 A1 WO 9316096A1
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Prior art keywords
gal
gene
dna
sequence
sequences
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PCT/US1993/001121
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English (en)
Inventor
Tai-Ping Sun
Howard M. Goodman
Frederick M. Ausubel
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The General Hospital Corporation
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Priority to AU36594/93A priority Critical patent/AU676468B2/en
Priority to EP93905822A priority patent/EP0626971A4/en
Priority to CA002129517A priority patent/CA2129517A1/fr
Priority to JP5514242A priority patent/JPH07503850A/ja
Publication of WO1993016096A1 publication Critical patent/WO1993016096A1/fr
Priority to KR1019940702835A priority patent/KR950700316A/ko

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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
    • C12N15/8297—Gibberellins; GA3
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the invention pertains to recombinant DNA technology. Specifically, 10 the invention relates to cDNA and genomic DNA corresponding to the GAl locus of Arabidopsis thaliana which encodes e/tf-kaurene synthetase, expression vectors containing such genes, hosts transformed with such vectors,
  • the regulatory regions of the GAl gene >. ⁇ the regulatory regions of the GAl gene, the use of such regulatory regions to direct the expression of operably-lin ed heterologous genes in transgenic 15 plants, the GAl protein substantially free of other A. thaliana proteins, antibodies capable of binding to the GAl protein, and to methods of assaying for the expression of the GAl gene and the presence of GAl protein in plant cells and tissues.
  • GAs Gibberellins
  • A. thaliana gal mutants are non-germinating, GA-responsive, male- sterile dwarfs whose phenotype can be converted to wild-type by repeated application of GA (Koornneef and van der Veen, Theor. Appl. Genet. 58:257- 263 (1980)). Koornneef et al.
  • the enzyme encoded by the GAl gene is involved in the conversion of geranylgeranyl pyrophosphate to «tf-kaurene (Barendse and Koornneef, Arabidopsis Inf. Serv. 19:25-28 (1982); Barendse et al., Physiol. Plant. 67:315-319 (1986); Zeevaart, J.A.D., in Plant Research '86, Annual Report of the MSU-DOE Plant Research Laboratory, 130-131 (East Lansing, MI, 1986)), a key intermediate in the biosynthesis of GAs (Graebe, J.E., Ann. Rev. Plant Physiol. 55:419-465 (1987)).
  • the GAl gene and other genes involved in GA biosynthesis is most likely caused by the unavailability of efficient transformation/ selection systems as well as the lack of available protein sequences.
  • a desired gene sequence is then identified by its capacity to complement (i.e. remedy) the genetic deficiencies of such mutant cells.
  • Such genetic identification permitted die genetic characterization of the gene sequences, and the construction of genetic maps which localized the gene sequence to a region of a particular chromosome (Taylor, Bacteriol. Rev. 34: 155 (1970)) .
  • clone i.e. to physically isolate
  • Random fragments of a genome could be introduced into self- replicating vectors to produce gene libraries, each of whose members contain a unique DNA fragment (Maniatis, T.
  • chromosome walking a desired sequence can be obtained by isolating a gene sequence which is capable of hybridizing to a particular reference sequence. This isolated gene sequence is then employed as a reference sequence in a subsequent hybridization experiment in order to clone a gene sequence which is adjacent to, and which partially overlaps, the originally isolated sequence. This newly isolated sequence will be physically closer to the desired gene sequence than was the originally isolated sequence. This process is repeated until the desired gene sequence has been obtained.
  • the ability to clone a gene sequence in the absence of genetic mutants or vectors, requires some initial information concerning the nucleotide sequence or restriction endonuclease digestion profile of the desired sequence.
  • the chromosome of a virus or cell can be characterized to produce a physical map based on either nucleotide sequence or restriction endonuclease cleavage data (i.e. an RFLP map). Using such a map, restriction fragments of the chromosome can be cloned without any prior determination as to their genetic function. More recently, gene cloning has been achieved by producing synthesizing oligonucleotide molecules whose sequence has been deduced from die amino acid sequence of an isolated protein, by forming cDNA copies of isolated RNA transcripts, by differential colony or library subtractive hybridizations using either two different RNA sources, or cDNA and RNA.
  • the competitive hybridization metiiod does not provide a large enough degree of enrichment. For example, enrichments of about one hundred fold were obtained for the sequences of interest in the above experiments. With enrichments of such low magnitude, the technique is practical only when dealing with large deletions. Indeed, even if the deletion covered 0.1 % of the genome, many putative positive clones have to be tested individually by labeling and probing genomic Southern blots (Southern, J., J. Molec. Biol. 95:503-517 (1975)). The method as it stands, then, is not practical for deletions on the order of 1 kbp (kilobasepair) unless one is dealing with a small prokaryotic genome.
  • Chromosome walking techniques may be used in other organisms to clone genetically defined loci if the mutant was obtained by transposon tagging, if the locus can be linked to markers in an RFLP map, or if an ordered library for the genome exists.
  • mutants with interesting phenotypes have been isolated but for which such procedures have not been developed, such as the GA synthesis mutants of A. thaliana.
  • many gene sequences cannot be isolated using the above methods.
  • chimeric plants produced through the use of these methods are known as either “chimeric” or “transgenic” plants.
  • a “chimeric” plant only some of the plant's cells contain and express the introduced gene sequence, whereas other cells remain unaltered. In contrast, all of the cells of a "transgenic” plant contain the introduced gene sequence.
  • Transgenic plants generally are generated from a transformed single plant cell. Many genera of plants have been regenerated from a single cell. (Friedt, W. etal Prog. Botany 49:192-215 (1987); Brunold, C. et al., Molec. Gen. Genet. 208:469-473 (1987); Durand, J. et al., Plant Sci. 62:263-272 (1989) which references are incorporated herein by reference).
  • chimeric and transgenic plants have substantial use as probes of natural gene expression.
  • the technologies When applied to food crops, the technologies have the potential of yielding improved food, fiber, etc.
  • Chimeric and transgenic plants having a specific temporal and spatial pattern of expression of the introduced gene sequence can be generated.
  • the expression of an introduced gene sequence can be controlled through the selection of regulatory sequences to direct transcription and or translation in a temporal or spatial fashion.
  • the invention is directed to isolated genomic DNA and cDNA corresponding to the GAl locus of A. thaliana, vectors containing such DNA, hosts transformed with such vectors, the regulatory regions that control the expression of the GAl protein, and the use of such regulatory sequences to direct the expression of a heterologous gene.
  • the invention further concerns the GAl protein, substantially free of other A. thaliana proteins, antibodies capable of binding the GAl protein, and the use of such GAl protein and antibodies thereto.
  • the invention further concerns chimeric and transgenic plants transformed witii the GAl encoding DNA sequence, or transformed with a heterologous gene controlled by the regulatory sequences of the GAl gene.
  • the invention further concerns the use of sequences encoding the GAl protein and antibodies capable of binding to die GAl protein to detect the expression of GAl and to isolate the regulatory proteins which bind to GAl gene sequences.
  • FIG. 1 A diagram of the enrichment and cloning method of the preferred embodiment of the present invention.
  • DNA is depicted as a solid line; biotinylated DNA is depicted as a striped black/white line; Sau3a adaptors are shown as an open line; avidin beads are shown as speckled circles; radiolabelled fragments are shown with asterisks.
  • Figure 2 Genetic and physical maps of the A. thaliana GAl locus.
  • A Physical map of the GAl region.
  • the heavy horizontal line is a Hindlll restriction map of the Landsberg erecta DNA encompassing the GA-1 locus. Hindlll restriction sites are depicted by vertical ticks extending below the horizontal line. The numbers immediately below the heavy horizontal line represent the size, in kilobase pairs, of the respective Hindlll restriction fragments. The location of the deletion in 31.89 is indicated by the hatched box.
  • the horizontal lines above the restriction map indicate the extent of the sequences contained in the ⁇ clone ⁇ GAl-3, the plasmid pGAl-2 (deposited January 7, 1993 pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms For The Purposes of Procedure (Budapest Treaty) with the American Type Culture Collection (ATCC) in Rockville, Maryland, U.S.A. 20852, and identified by ATCC Accession No.
  • Figure 3 Detection of deletions and insertions in 31.89 and 6.59 DNA, respectively. Autoradiograms are shown for Southern blots probed with (A) the 250 bp Sau3A fragment from pGAl-1 (see Example 1), and (B) the 6 kb fragment from pGAl-2 (ATCC No. 75394) that covers the entire deleted region in 31.89 ( Figure IB). Both blots A and B contain Ht ' /ttflll-digested DNA isolated from Landsberg erecta (lane 1), and three ga-1 mutants, 31.89 (lane 2), 29.9 (lane 3), and 6.59 (lane 4). The arrows in panel B indicate altered Hindlll fragments in 31.89 (4.2 kb) and 6.59 (1.3 and 3.3 kb). Figure 4. Photograph and Southern blots of wild-type and transgenic plants containing GAl gene.
  • FIG. 1 Photograph of six-week-old A. thaliana Landsberg erecta plants. Left to right: a ga-1 mutant (31.89), a transgenic ga-1 mutant (31.89) plant containing the 20 kb insert from pGAl-4 (ATCC No. 75395), a wild-type Landsberg erecta plant. Autoradiograms are shown for Southern blots probed with (B) the 6 kb fragment from pGAl-2 (ATCC No. 75394), and (C) pOCAl ⁇ DNA which is the vector for pGAl-4 (ATCC No. 75395) (see Figure 2).
  • Blots B and C contain H tuftH-digested DNA from Landsberg erecta (lane 1 in B), Columbia (lane 2 in B, lane 1 in C), 31.89 (lane 3 in B, lane 2 in C), and two T3 generation transgenic ga-1 (31.89) plants transformed with pGAl-4 (ATCC No. 75395) (lane 4,5 in B; lane 3,4 in C).
  • RNA was from wild type four-week-old plants (lane 1), five-week-old wild type plants, (lane 2), immature wild-type siliques (lane 3), and four week-old ga-1 mutant 31.89 plants (lane 4).
  • FIG. 6 Partial cDNA sequence of the GAl gene (Sequence ID No. 1).
  • the GAl DNA strand complementary to GAl mRNA is shown in a 5 '-3' orientation.
  • the GAl variant d352 has the identical sequence to that shown except for the substitution of an A for the G at position 425.
  • the GAl variant A428 has the identical sequence to that shown except for the substitution of a T for the C at position 420.
  • the GAl variant Bo27 has the identical sequence to that shown except for the substitution of a T for the C at position 246.
  • Figure 7. Partial cDNA sequence of the GAl gene (Sequence ID No. 2).
  • the GAl DNA strand shown is analogous to GAl mRNA and complementary to the strand shown in Figure 6.
  • the GAl variant d352 has the identical sequence to that shown except for the substitution of a T for the C at position 479.
  • the GAl variant A428 has the identical sequence to that shown except for the substitution of an A for the G at position 484.
  • the GAl variant Bo27 has the identical sequence to that shown except for the substitution of an A for the G at position 658.
  • Genomic subtraction is a method for enriching, and clonally isolating a gene sequence present in one nucleic acid population but absent in another. Following the procedures outlined herein that demonstrate the cloning of the GAl gene, it is now also possible to isolate other genes involved in GA syntiiesis.
  • vectors containing genomic or cDNA encoding die GAl protein (Sequence ID No. 1), or a fragment thereof, are provided. Specifically, such vectors are capable of generating large quantities of the GAl sequence, substantially free of other A. thaliana DNA.
  • Vectors for propagating a given sequence in a variety of host systems are well known and can readily be altered by one of skill in the art such that the vector will contain the GAl sequence and will be propagated in a desired host.
  • Such vectors include plasmids and viruses and such hosts include eukaryotic organisms and cells, for example yeast, insect, plant, mouse or human cells, and prokaryotic organisms, for example E. coli and B. sutillus.
  • a sequence is said to be “substantially free of other A. thaliana DNA” when the only A. thaliana DNA present in the sample or vector is of a specific sequence.
  • a "DNA construct" refers to a recombinant, man-made
  • a fragment thereof relates to any polynucleotide subset of the entire GAl sequence.
  • the most preferred fragments are those containing the active site of the enzyme encoded by GAl, or the regulatory regions of the GAl protein and gene respectively.
  • expression vectors are described which are capable of expressing and producing large quantities of the GAl protein, substantially free of other A. thaliana proteins.
  • a protein is said to be "substantially free of other A. thaliana proteins" when the only A. thaliana protein present in the sample is the expressed protein. Though proteins may be present in the sample which are homologous to other A. thaliana proteins, the sample is still said to be substantially free as long as the homologous proteins contained in the sample are not expressed from genes obtained from A. thaliana.
  • a nucleic acid molecule, such as DNA is said to be “capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked" to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • the precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of gene synthesis.
  • Such regions will normally include those 5 '-non- coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and die like. If desired, the non-coding region 3 ' to the gene sequence coding for the
  • GAl gene may be obtained by die above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3 '-region naturally contiguous to the DNA sequence coding for the GAl gene, the transcriptional termination signals may be provided. Where me transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if die nature of the linkage between the two DNA sequences does not (1) result in die introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the GAl gene sequence, or (3) interfere with the ability of the GAl gene sequence to be transcribed by die promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the GAl gene protein (or a functional derivative tiiereof) in either prokaryotic or eukaryotic cells.
  • Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E.
  • coU W3110 F, lambda " - prototrophic (ATCC 27325)
  • otiier enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species.
  • the GAl gene will not be glycosylated.
  • the procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • the GAl gene (or a functional derivative tiiereof) in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary to operably link the GAl gene encoding sequence to a functional prokaryotic promoter.
  • a prokaryotic promoter such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters examples include die int promoter of bacteriophage ⁇ , the bla promoter of the 3-lactamase gene sequence of pBR322, and die CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, etc.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, lacZ, lad, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen, I., et al, J. Bacteriol. 162:176-182 (1985)) and the sigma-28-specif ⁇ c promoters of B.
  • subtilis (Gilman, M.Z., et al, Gene 52:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: TThe Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al, Mol. Gen. Genet. 205:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick, B.R. , (J. Ind. Microbiol i:277-282 (1987)); Cenatiempo, Y. (Biochimie 65:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 75:415-442 (1984)).
  • ribosome binding sites are disclosed, for example, by Gold, L., et al (Ann. Rev. Microbiol. 55:365-404 (1981)).
  • Preferred eukaryotic hosts include yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture.
  • Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/O-AG14 or me myeloma P3x63Sg8, and their derivatives.
  • Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.
  • IMR 332 neuroblastoma cell lines
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, etc., may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • Yeast provides substantial advantages in that it can also carry out post- translational peptide modifications.
  • Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • Any of a series of yeast gene sequence expression systems incorporating promoter and termination elements from the actively expressed gene sequences coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene sequence can be utilized.
  • Another preferred host is insect cells, for example the Drosophila larvae.
  • insect cells for example the Drosophila larvae.
  • the Drosophila alcohol dehydrogenase promoter can be used. Rubin, G.M., Science 240:1453-1459 (1988).
  • baculovirus vectors can be engineered to express large amounts of the GAl gene in insects cells (Jasny, B.R., Science 255:1653 (1987); Miller, D.W., etal, in Genetic Engineering (1986), Setlow, J.K., etal, eds., Plenum, Vol. 8, pp. 277-297).
  • eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include die promoter of the mouse metallothionein I gene sequence (Hamer, D. , et al. , J. Mol Appl. Gen.
  • the GAl gene encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, die expression of the GAl gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of die introduced sequence into the host chromosome. In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as tran ⁇ scription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama, H., Molec.
  • die introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: die ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whetiier it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E.
  • coli such as, for example, pBR322, Col ⁇ l, pSClOl, pACYC 184, xVX.
  • plasmids are, for example, disclosed by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomyces plasmids include pIJlOl (Kendall, K.J., et al, J. Bacteriol 169:4177-4183 (1987)), and streptomyces bacteriophages such as C31 (Chater, K.F., et al , In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J.F., et al (Rev. Infect. Dis. 5:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 55:729-742 (1978)).
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, D., etal, Miami Wntr. Symp. 19:265-274 (1982); Broach, J.R. , In: TThe Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 25:203-204 (1982); Bollon, D.P., et al., J. Clin. Hematol. Oncol. 20:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene sequence Expression, Academic Press, NY, pp. 563-608 (1980)).
  • the DNA constructs may be introduced into an appropriate host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene sequence(s) results in the production of die GAl gene, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • the GAl protein can be readily isolated using standard techniques such as immunochromatography or HPLC to produce GAl protein free of other A. thaliana proteins.
  • chromosomal walking techniques By employing chromosomal walking techniques, one skilled in the art can readily isolate other full length genomic copies of GAl as well as clones containing the regulatory sequences 5' of the GAl coding region.
  • full length genomic copies refers to a DNA segment which contains a protein's entire coding region.
  • regulatory sequences refers to DNA sequences which are capable of directing the transcription and/or translation of an operably linked DNA sequence.
  • Such regulatory sequences may include, but are not limited to, a promoter, ribosome binding site, and regulatory protein binding site.
  • One skilled in the art can readily identify certain regulatory sequences by comparing sequences found 5' to a coding region with known regulatory sequence motifs, such as those recognized by the computer programs "motif" and "consensus”.
  • die GAl DNA sequences disclosed herein were used to screen an A. thaliana genomic DNA library via chromosome walking. Genomic DNA libraries for A.
  • thaliana are commercially available (Clontech Laboratories Inc, and American Type Culture Collection) or can be generated using a variety of techniques known in the art. (Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)). By isolating clones which overlap and occur 5' or 3' to the sequences disclosed herein, sequences hybridizing to Sequence ID No. 1 were identified and isolated. Such sequences are contained in die vectors PGA1-4 (ATCC No. 75395) and ⁇ GAl-3. Regulatory sequences are those which occur 5' to a coding region.
  • the preferred regulatory sequences of the present invention are those which appear from about -2 kb - 0 bp 5' of the GAl starting codon (AGT/Met). The more preferred sequences appear from about -500 bp -0 bp, the most preferred being sequences from about -250 bp - 0 bp.
  • functional derivatives of the GAl gene as well as the regulatory sequence of this gene. Such derivatives allow one skilled in the art to associate a given biological activity with a specific sequence and/or structure and tiien design and generate derivatives with an altered biological or physical property.
  • the preparation of a functional derivative of the GAl sequence can be achieved by site-directed mutagenesis.
  • Site-directed mutagenesis allows the production of a functional derivative through the use of a specific oligonucleotide which contains the desired mutated DNA sequence.
  • d e mutation perse need not be predetermined.
  • random mutagenesis may be conducted at a target region and die newly generated sequences can be screened for the optimal combination of desired activity.
  • the functional derivatives created this way may exhibit die same qualitative biological activity as die naturally occurring sequence when operably linked to a heterologous gene.
  • the derivative may however, differ substantially in such characteristics as to the level of induction in response to phytohormones.
  • a functional derivative made by site-directed mutagenesis can be operably linked to a reporter gene, such as /J-glucuronidase (GUS), and die chimeric gene can men be quantitatively-screened for phytohormone responsiveness in chimeric or transgenic plants, or in a transient expression system.
  • GUS J-glucuronidase
  • antibodies which bind die GAl protein are provided.
  • an antibody which binds to the GAl protein can be generated in a variety of ways using techniques known in the art. Specifically, in one such method, GAl protein purified from either an expression host or from plant tissue is used to immunize a suitable mammalian host. One skilled in the art will readily adapt known procedures in order to generate both polyclonal and monoclonal an ⁇ -GAl antibodies. (Harlow, Antibodies, Cold Spring Harbor Press (1989)).
  • anti-G-47 antibodies can be generated using synthetic peptides. Using die deduced amino acid sequence encoded by the GAl gene described herein, a synthetic peptide can be made, such tiiat when administered to an appropriate host, antibodies will be generated which bind to the GAl protein.
  • a procedure is described for detecting die expression of the GAl gene or the presence of the GAl protein in a cell or tissue.
  • RNA encoding GAl or the GAl protein itself.
  • assay formats such as in situ hybridization, ELISA, and protein or nucleic acid blotting techniques, in order to detect the presence of RNA encoding GAl, or the GAl protein itself. Utilizing such a detection system, it is now possible to identify the specific tissues and cells which transcribe or translate the GAl gene.
  • a method for creating a chimeric or transgenic plant in which the plant contains one or more exogenously supplied genes which are expressed in the same temporal and spatial manner as GAl.
  • a chimeric or transgenic plant is generated such that it contains an exogenously supplied expression module.
  • the expression module comprises the regulatory elements of the GAl gene, operably linked to a heterologous gene.
  • the regulatory region of die GAl gene is contained in die region from about -2 kb to 0 bp, 5' to the GAl start codon (Met).
  • One skilled in the art can readily generate expression modules containing this region, or a fragment thereof.
  • RNA encoding GAl in another embodiment, a method of modulating the translation of RNA encoding GAl in a chimeric or transgenic plant is described.
  • modulation entails the enhancement or reduction of die naturally occurring levels of translation.
  • the translation of GAl encoding RNA can be reduced using the technique of antisense cloning.
  • Antisense cloning has been demonstrated to be effective in plant systems and can be readily adapted by one of ordinary skill to utilize the GAl gene. (Oeller et al. , Science 254:437-
  • antisense cloning entails the generation of an expression module which encodes an RNA complementary (antisense) to the RNA encoding GAl (sense).
  • antisense RNA By expressing the antisense RNA in a cell which expresses the sense strand, hybridization between the two RNA species will occur resulting in the blocking of translation.
  • the activity of GAl can be suppressed in a transgenic or chimeric plant by transforming a plant with an expression module which encodes an an ⁇ -GAl antibody.
  • the expressed antibody will bind the free GAl and tiius impair the proteins ability to function.
  • DNA encoding an an -GAl antibody can readily be obtained using techniques known in the art. In general, such DNA is obtained as cDNA, generated from mRNA which has been isolated from a hybridoma producing anti-G__7 antibodies. Metiiods of obtaining such a hybridoma are described earlier.
  • Procedures for the isolation of regulatory factors capable of binding to a specific DNA sequence are well known in the art.
  • One such method is affinity chromatography.
  • DNA containing the regulatory sequence is immobilized on an appropriate matrix, such as Sepharose, and used as an affinity matrix in chromatography (Arcangioli B. , et al, Eur. J. Biochem. 779:359-364 (1989)).
  • Proteins which bind the GAl regulatory element can be extracted from plant tissues expressing die GAl gene.
  • a protein extract obtained in such a fashion is applied to a column which contains immobilized GAl regulatory region. Proteins which do not bind to the DNA sequence are washed off the column and proteins which bind to die DNA sequence are removed from the column using a salt gradient.
  • the DNA binding protein obtained this way can be further purified using procedures such as ion exchange chromatography, high performance liquid chromatography, and size exclusion chromatography.
  • die protein can be readily assayed using a gel retardation assay (Garner, M.M. et al, Nucl. Acid Res. 9:3047 (1981) and Fried, M. et al., Nucl Acid Res. 9:6506 (1981)).
  • a partial amino acid sequence can be obtained from die N-terminal of the protein.
  • the protein can be tryptically mapped and die amino acid sequence of one of the fragments can be determined.
  • the deduced amino acid sequence is used to generate an oligonucleotide probe.
  • the probe's sequence can be based on codons which are known to be more frequently used by the organism (codon preference), or, alternatively, die probe can consist of a mixture of all the possible codon combination which could encode the polypeptide (degenerate).
  • Such a probe can be used to screen either a cDNA or genomic library for sequences which encode die DNA binding protein.
  • the sequence of the DNA encoding the binding protein can be determined, the gene can be used to obtain large amounts of the protein using an expression system, or in mutational analysis can be performed to further define the functional regions within the protein which interacts with the DNA.
  • proteins which bind to the GAl regulatory elements can be isolated by identifying a clone expressing such a protein using the technique of Southeastern blotting (Sharp, Z.D. etal, Biochim Biophys Acta, 1048:306- 309 (1990), Gunther, C. V. etal, Genes Dev.
  • a labeled DNA sequence is used to screen a cDNA expression library whose expressed proteins have been immobilized on a filter via colony or plaque transfer.
  • the labeled DNA sequences will bind to colonies or plaques which express a protein capable of binding to the particular DNA sequence.
  • Clones expressing a protein which binds to the labeled DNA sequence can be purified and the cDNA insert which encodes the DNA binding protein can be isolated and sequenced.
  • the isolated DNA can be used to express large amounts of the protein for further purification and study, to isolate the genomic sequences corresponding to d e cDNA, or to generate functional derivative of die binding protein.
  • Genomic subtraction between A. thaliana Landsberg erecta DNA and gal 31.89 DNA was performed as described previously (Straus and Ausubel, Proc. Natl. Acad. Sci. USA 57:1889-1893 (1990)) with the following modifications.
  • A. thaliana Landsberg erecta DNA and gal mutant (31.89) DNA were isolated and purified by CsCl gradient centrifugation as described (Ausubel et al, in Current Protocols in Molecular Biology, Vol. 1 (Greene Publishing Associates/Wiley-Interscience, New York, 1990)).
  • 0.25 ⁇ g of Landsberg erecta DNA digested witii Sau3A was hybridized witii 12.5 of the gal mutant 31.89 DNA that had been sheared and photobiotinylated. 10 ⁇ g of biotinylated 31.89 DNA was added in each additional cycle.
  • Hybridizations were carried out for at least 20 hours at a concentration of 3 ⁇ g DNA/ ⁇ l at 65 °C. After five cycles of subtraction, the amplified products were ligated to Sau3A adaptors, amplified by PCR and ligated into the Smal site of pUC 13. After five cycles of subtractive hybridization, the remaining DNA fragments were enriched for sequences present in wild-type DNA but missing from 31.89 DNA. These DNA fragments were amplified by the polymerase chain reaction (PCR) and cloned. One of six clones examined (pGAl-1) contained a 250 bp Sau3A fragment that was deleted from 31.89 DNA.
  • PCR polymerase chain reaction
  • pGAl-1 DNA was used as a hybridization probe to isolate larger genomic fragments corresponding to the deletion in 31.89. These cloned fragments are shown in Figure 2B.
  • ⁇ GAl-3 was isolated from a Landsberg erecta genomic library constructed in ⁇ FIX (Voytas et al, Genetics 126:713-721 (1990)) using 32 P- labelled pGAl-1 as probe.
  • pGAl-2 (ATCC No. 75394) was obtained by ligating a 6 kb Sall-EcoRl fragment from ⁇ GAl-3 into the Xhol and Ec ⁇ RI sites of pBluescriptll SK (Stratagene).
  • pGAl-4 (ATCC No. 75395) was isolated from a genomic library of A. thaliana ecotype Columbia DNA constructed in the binary vector pOCAl ⁇ (Olszewski et al, Nucl Acid Res. 76:10765-10782 (1988)) which contains the T-DNA borders required for efficient transfer of cloned DNA into plant genomes (Olszewski et al. , Nucl. Acid Res. 76:10765-10782 (1988)). Plasmid pGAl-2 (ATCC No.
  • Agrobacterium tumefaciens strain LBA4404 containing pGAl-4 was used to infect root explants of gal mutant 31.89 and kanamycin-resistant (Km 1 ) transgenic plants were selected as described (Valvekens et al, Proc. Natl. Acad. Sci. USA 65:5536-5540 (1988)). 130 Km 1 plants were regenerated which set seeds in the absence of exogenous GA (Tl generation). 50 to 300 seeds from each of 4 different Tl plants showed 100% linkage of the gal and K ⁇ phenotypes which segregated approximately 3: 1 to the g..7/Km s phenotype (T2 generation).
  • Seeds of transgenic gal and wild-type plants were germinated on agarose plates containing IX Murashige & Skoog salts and 2% sucrose with or without kanamycin (MS plates) . Seeds of the gal mutant 31.89 were soaked in 100 ⁇ M GA 3 for 4 days before being germinated on MS plates. Seven-day- old seedlings were transferred to soil.
  • mutants Bo27, A428, and d352 are PCR artifacts or are due to die highly polymorphic nature of the GAl locus because die 1.2 kb Hindlll fragment amplified and sequenced from mutants NG4 and NG5 both had the wild-type sequence. Moreover, the PCR products were sequenced directly and die sequence analysis was carried out twice using the products of two independent amplifications for each allele examined.
  • RNA of each sample was size-fractionated on a 1 % agarose gel (Maniatis et al. , in Molecular Cloning: A Laboratory Manual, 197-201 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982)), transferred to
  • the linkage map of A. thaliana is approximately 600 cM and the genome size is approximately 1.08 x 10 8 bp (Goodman et at., unpublished results). This is equivalent to approximately 6 x 10 " * cM per base pair, in good agreement with the observed recombination frequency in the GAl locus.
  • thaliana at the chl-3 (Wilkinson and Crawford, Plant Cell 5:461-471 (1991)), tt-3 (B. Shirley and H. M. Goodman, unpublished result) and gl-1 loci (D. Marks, personal communication).
  • An expression vector is constructed as previously described such that it expresses an RNA complementary to the sense strand GAl RNA.
  • the antisense GAl RNA is expressed in a constitutive fashion using promoters which are constitutiveiy expressed in a given host plant, for example, the cauliflower mosaic virus 35S promoter.
  • the antisense RNA is expressed in a tissue specific fashion using tissue specific promoters. As described earlier, such promoters are well known in the art.
  • the antisense construct pPO35 (Oeller et al , Science 254:437-439 (1991)) is cut with BamHl and SAC1 to remove the tACC2 cDNA sequence. After removing the tACC2 cDNA, the vector is treated with the Klenow fragment of E. coli DNA polymerase I to fill in the ends, and the sequence described in S ⁇ Q ID. NO. 1 is blunt end ligated into the vector. The ligated vector is used to transform an appropriate E. coli strain.
  • Colonies containing the ligated vector are screened using colony hybridization or Southern blotting to obtain vectors which contain the GAl cDNA in die orientation which will produce antisense RNA when transcribed from the 35S promoter contained in the vector.
  • the antisense GAl vector is isolated from a colony identified as having the proper orientation and die DNA is introduced into plant cells by one of the techniques described earlier, for example, electroporation or Agrobacterium/Ti plasmid mediated transformation.
  • Plants regenerated from the transformed cells express antisense GAl RNA.
  • the expressed antisense GAl RNA binds to sense strand GAl RNA and thus prevent translation.
  • ADDRESSEE Sterne, Kes ⁇ ler, Goldstein & Fox
  • NAME Cimbala, Michele A.
  • CTGCAGGAAT TCCTTTTTTTTT TTTTTTTTTTTT TGGCTTTGAG TGAAGTACAT AGGACCCATC 60
  • GTAGCATTCC CATCGTTGCT TGAGATAGCT CGAGGAATAA ACATTGATGT ACCGTACGAT 720

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Abstract

L'invention se rapporte au clonage et à la mise en séquence d'ADN correspondant aux sites de GA1 de A. thaliana codant la synthétase de ent-kaurene, ainsi que des vecteurs contenant ledit ADN, à des vecteurs capable d'exprimer ledit ADN, ainsi qu'à des hôtes transformés au moyen desdits vecteurs. L'invention se rapporte, de plus, à l'utilisation du gène GA1, ainsi qu'à des régions régulatoires dudit gène, dans la production de plantes chimères et transgéniques.
PCT/US1993/001121 1992-02-18 1993-02-05 Adn de gibberelline recombinante et utilisations WO1993016096A1 (fr)

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AU36594/93A AU676468B2 (en) 1992-02-18 1993-02-05 Recombinant gibberellin DNA and uses thereof
EP93905822A EP0626971A4 (en) 1992-02-18 1993-02-05 Recombinant gibberellin dna and uses thereof.
CA002129517A CA2129517A1 (fr) 1992-02-18 1993-02-05 Adn gibberelline recombinant, et son usage
JP5514242A JPH07503850A (ja) 1992-02-18 1993-02-05 組み替えジベレリンｄｎａおよびその用途
KR1019940702835A KR950700316A (ko) 1992-02-18 1994-08-17 재조합 지베렐린 디엔에이(dna) 및 이들의 사용 방법(recombinant gibberellin dna and uses thereof)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995035383A3 (fr) * 1994-06-17 1996-02-15 Pioneer Hi Bred Int Genes de plantes affectant la biosynthese de l'acide gibberellique
WO1996005317A1 (fr) * 1994-08-16 1996-02-22 The General Hospital Corporation Adn ga4, proteine codee et utilisation
EP0692537A3 (fr) * 1994-06-17 1996-10-23 Massachusetts Gen Hospital ADN récombinante codant pour gibberellin et son utilisation
US5612191A (en) * 1994-06-17 1997-03-18 Pioneer Hi-Bred International, Inc. Plant genes affecting gibberellic acid biosynthesis
EP0768381A2 (fr) * 1995-10-09 1997-04-16 The Institute Of Physical & Chemical Research Kaurene-Synthase
EP0798387A1 (fr) * 1994-09-30 1997-10-01 Kabushiki Kaisha Toyota Chuo Kenkyusho Procédé de détection d'acides nucléiques
WO1997041240A1 (fr) * 1996-05-01 1997-11-06 Pioneer Hi-Bred International, Inc. Procedes et compositions transgeniques pour la production de legumes et de fruits parthenocarpiques
US5912415A (en) * 1996-05-16 1999-06-15 Regents Of The University Of Minnesota Arabidopsis spindly gene, methods of identification and use
WO2000009722A3 (fr) * 1998-08-10 2000-09-28 Monsanto Co Methode de regularisation du taux de gibberelline
US10323256B2 (en) 2011-12-09 2019-06-18 Ceres, Inc. Transgenic plants having altered biomass composition

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897345A (en) * 1987-07-20 1990-01-30 Lubrizol Genetics Inc. Pollen tube growth assay

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4897345A (en) * 1987-07-20 1990-01-30 Lubrizol Genetics Inc. Pollen tube growth assay

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FEBS Letters, Vol. 307, No. 1, issued July 1992, HIATT et al., "Monoclonal Antibody Engineering in Plants", pages 71-75, see page 73. *
Nucleic Acids Research, Vol. 17, No. 18, issued 1989, CORNELISSEN, "Nuclear and Cytoplasmic Sites for Anti-Sense Control", pages 7203-7209, see the entire document. *
Plant Cell, Vol. 4, No. 2, issued 1992, SUN et al., "Cloning the Arabidopsis GA-1 Locus by Genomic Subtraction", see Abstract. *
See also references of EP0626971A4 *
The EMBO Journal, Vol. 9, No. 11, issued 1990, SCHINDLER et al., "Photoregulated Gene Expression May Involve Ubiquitous DNA Binding Proteins", pages 3415-3427, see the Abstract. *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866779A (en) * 1992-02-18 1999-02-02 The General Hospital Corporation Recombinant gibberellin DNA and uses thereof
US5773288A (en) * 1994-06-17 1998-06-30 Pioneer Hi-Bred International, Inc. Plant genes affecting gibberellic acid biosynthesis
EP0692537A3 (fr) * 1994-06-17 1996-10-23 Massachusetts Gen Hospital ADN récombinante codant pour gibberellin et son utilisation
US5612191A (en) * 1994-06-17 1997-03-18 Pioneer Hi-Bred International, Inc. Plant genes affecting gibberellic acid biosynthesis
US5767375A (en) * 1994-06-17 1998-06-16 Pioneer Hi-Bred International, Inc. Plant genes affecting gibberellic acid biosynthesis
WO1995035383A3 (fr) * 1994-06-17 1996-02-15 Pioneer Hi Bred Int Genes de plantes affectant la biosynthese de l'acide gibberellique
US5925807A (en) * 1994-08-16 1999-07-20 General Hospital Corporation GA4 DNA, protein and method of use
WO1996005317A1 (fr) * 1994-08-16 1996-02-22 The General Hospital Corporation Adn ga4, proteine codee et utilisation
US6013472A (en) * 1994-08-16 2000-01-11 The General Hospital Corporation GA4 DNA, protein and methods of use
EP0798387A1 (fr) * 1994-09-30 1997-10-01 Kabushiki Kaisha Toyota Chuo Kenkyusho Procédé de détection d'acides nucléiques
US5728531A (en) * 1994-09-30 1998-03-17 Kabushiki Kaisha Toyota Chuo Kenkyusho Method of detecting nucleic acid
EP0768381A2 (fr) * 1995-10-09 1997-04-16 The Institute Of Physical & Chemical Research Kaurene-Synthase
US6020176A (en) * 1995-10-09 2000-02-01 The Institute Of Physical And Chemical Research Kaurene synthase
EP0768381A3 (fr) * 1995-10-09 1999-01-27 The Institute Of Physical & Chemical Research Kaurene-Synthase
US5877400A (en) * 1996-05-01 1999-03-02 Pioneer Hi-Bred International, Inc. Transgenic methods and compositions for producing parthenocarpic fruits and vegetables
WO1997041240A1 (fr) * 1996-05-01 1997-11-06 Pioneer Hi-Bred International, Inc. Procedes et compositions transgeniques pour la production de legumes et de fruits parthenocarpiques
US5912415A (en) * 1996-05-16 1999-06-15 Regents Of The University Of Minnesota Arabidopsis spindly gene, methods of identification and use
WO2000009722A3 (fr) * 1998-08-10 2000-09-28 Monsanto Co Methode de regularisation du taux de gibberelline
US6723897B2 (en) 1998-08-10 2004-04-20 Monsanto Technology, Llc Methods for controlling gibberellin levels
US7195917B2 (en) 1998-08-10 2007-03-27 Monsanto Technology Llc Methods for controlling gibberellin levels
US10323256B2 (en) 2011-12-09 2019-06-18 Ceres, Inc. Transgenic plants having altered biomass composition
US10815496B2 (en) 2011-12-09 2020-10-27 Ceres, Inc. Transgenic plants having altered biomass composition
US10822616B2 (en) 2011-12-09 2020-11-03 Ceres, Inc. Transgenic plants having altered biomass composition
US11299747B2 (en) 2011-12-09 2022-04-12 Ceres, Inc. Transgenic plants having altered biomass composition

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