EP0928338A1 - Procede de production de plantes a sterilite femelle - Google Patents

Procede de production de plantes a sterilite femelle

Info

Publication number
EP0928338A1
EP0928338A1 EP97909268A EP97909268A EP0928338A1 EP 0928338 A1 EP0928338 A1 EP 0928338A1 EP 97909268 A EP97909268 A EP 97909268A EP 97909268 A EP97909268 A EP 97909268A EP 0928338 A1 EP0928338 A1 EP 0928338A1
Authority
EP
European Patent Office
Prior art keywords
ptc
gene
acetyl
plants
ptt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP97909268A
Other languages
German (de)
English (en)
Inventor
Klaus Bartsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience AG
Original Assignee
Hoechst Schering Agrevo GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=7806895&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP0928338(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Hoechst Schering Agrevo GmbH filed Critical Hoechst Schering Agrevo GmbH
Publication of EP0928338A1 publication Critical patent/EP0928338A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/829—Female sterility
    • 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
    • C12N9/14—Hydrolases (3)

Definitions

  • the invention relates to the use of deacetylase genes for the production of transgenic plants using tissue-specific promoters. The development of certain parts of plants can be specifically prevented in these plants.
  • PTC Phosphinothricin
  • GS glutamine synthetase
  • PTC is a "building block" of the antibiotic phosphinothricyl-alanyl-alanine.
  • This tripeptide (PTT) is active against Gram-positive and Gram-negative bacteria and also against the fungus Botrytis cinerea.
  • PTT is produced by the strain Streptomyces viridochromogenes TÜ494, which is deposited and available from the German Collection for Microorganisms under the numbers DSM 40736 and DSM 41 12. From German Patent 2,717,440 it is known that PTC acts as a total herbicide.
  • the published application (EP-A-0257542) describes how to prepare herbicide-resistant plants using a phosphinothricin-N-acetyltansferase (pat) gene.
  • the phosphinothricin-N-acetyltransferase encoded by the pat gene modifies the intracellular PTC and detoxifies the herbicide.
  • the present invention now describes the use of deacetylase genes (dea), the expression products of which intracellularly N-acetyl-phosphinothricin (N-Ac-PTC) or w. Can deacetylate N-Ac-PTT and thus make it antibiotically active again for the production of sterile plants.
  • deacetylase genes deacetylase genes
  • N-acetylphosphinothricin tripeptide deacetylase gene can be isolated from S. viridochromogenes TÜ494.
  • the dea gene is located downstream of the pat gene on the already known 4.0 kb BamHI fragment (EP-A-0 257 542). This gene is located on a BglII-BamHI fragment and is precise in its sequence determined (Fig. 1 and Tab 1)
  • the protein sequence is defined by the DNA sequence
  • the phosphinoth ⁇ cin-N-acetyltransfera e encoded by the pat gene actually serves for acetylation of desmethyl-PTC in PTT biosynthesis and, because of its unspecificity, can detoxify PTC
  • an insufficiently specific N-acetyl-PTT-deacetylase can now be used to activate N-acetyl-phosphinoth ⁇ cin
  • the pat gene in low copy number in E coli is unable to confer PTT resistance, since the endogenous deacetylase is the effect of phosphinothricm-N-acetyltransferase
  • this deacetylase activity can be detected directly by the effective inhibition of the GS activity after the addition of N-acetylphosphinoth ⁇ cin.
  • N-Ac-PTC is converted to PTC by the deacetylase, which is then converted into b known to inhibit GS, which can be seen in the ⁇ -glutamyl transferase assay (Bender et al, J Bactenol 129, 1001- 1009, 1977) This is due to an endogenous deacetylase activity of
  • the deacetylase gene from E coli can thus be obtained by creating a gene bank in, for example, the E coli argE mutant or in a newly isolated mutant using conventional methods (Maniatis et al, Molecular Clonmg a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982)
  • pat and dea genes can be used together with tissue-specific promoters in order to specifically prevent the development of certain plant tissues.
  • a special application is, for example, the production of sterile plants
  • RNAse gene is fused with a tapetum-specific promoter (Ma ⁇ ani et al .; Nature 347, 737-741, 1990).
  • the exclusive expression of the gene in the tapetum cells ensures the selective destruction of the tissue and thus prevents the formation of mature pollen.
  • a plant that carries this gene should only be able to form seeds after cross-fertilization
  • the invention therefore includes tissue-specific inhibition with the aid of a deacetylase gene
  • Pat activity by PTT or PTC-resistant plants is used to transform with a deacetylase gene under the control of the tissue-specific active promoter in plants. After application of PTT or PTC, expression of Deacetylase gene to abolish the phosphinoth ⁇ cin-N-acetyltransferase activity in the corresponding tissues. These are then selectively killed while the rest of the plant is resistant
  • This system can be simplified by using N-acetyl-phosphinoth ⁇ cin or N-acetyl-phosphinothncin-Tn peptide. Both substances are not active as herbicides, but are taken up by plants, transported and not immediately broken down. Deacetylase activity for N-acetyl-phosphinothncin and N - Acetyl-phosphinoth ⁇ cin-T ⁇ peptide has not yet been detected in plants. Thus, the 2-gene system described above can be reduced to a 1-gene system and thus decisively simplified, as explained further below. Any plants can be controlled with a streptomycete deacetylase gene of a tissue-specific promoter are transformed. After application of N-acetyl-phosphinoth ⁇ cin or N-acetyl-phosphinoth ⁇ cin-t ⁇ peptide, the tissue-specific expression leads to the immediate death of the corresponding tissue
  • tissue-specific promoters can be used as tissue-specific promoters, the selective expression of which has been demonstrated in certain tissues, preferably the female organs.
  • female organs includes gametophytes and him surrounding or neighboring tissues, such as gynoceum (fruit leaves), ovules, placenta, pestle (fruit nodes, pistil, scar)
  • Robert et al describe stigma-specific promoters from rapeseed (Robert et al, 1994), Sato et al Pistil-specific Promote were also described (Sato et al, 1991, Dzelzkalns et al, 1993, WO 94/25613)
  • promoters are also suitable for the process according to the invention which, although not specifically active in the female organs, are nevertheless expressed in a tissue which is essential for the development of the functional blood, embryo and semen
  • promoters which are subject to a different type of regulation (for example temporally, stress-related, environmentally dependent) and which occurs in a tissue-specific manner
  • the method can preferably be used for the production of sterile plants
  • Example 1 Fusion of the deacetylase coding region with eukaryotic transcription signals
  • the plasmid pPRI (see EP-0 257 542) was isolated from an E coli strain and digested with BamHI and BglII. The digested DNA was separated in an agarose gel and a 0 9 kb fragment was isolated from the gel.
  • the vector pROKI (Baulcombe et al, Nature 321, 446-449, 1986) was also restricted with BamHI. The two approaches were combined and ligated.
  • the ligation mixture was prepared according to E. coli S17 1 (Simon et al, Bio / Technology 1 784-791 1983) transformed Colonies growing on media containing kanamycin were transferred to nitrocellulose filters and lysed after 12 hours of incubation at 37 ° C.
  • the DNA of the bacteria was fixed on the filter.
  • the 0.9 kb fragment isolated from the agarose gel was made single-stranded by incubation at 100 ° C. Then the missing one was removed Strand synthesized with Klenow polymerase and digoxigenin-labeled nucleotides. The labeled strand was used as a sample for hybridization with the bacterial DNA bound to the filter. Hybridizing clones were detected using an antibody reaction.
  • the DNA of the positive clones was isolated by Qiagen lysis and with BamHI / EcoRI and BamHI / HindIII digested. This restriction enables the orientation of the inserted 0 9 kb fragment to be determined.
  • the plasmid with the orientation I was designated as plB17 1, that with the orientation II as plB17 2 (see FIG. 2)
  • Example 2 Detection of the deacetylation of N-acetyl-PTC and N-acetyl-PTT by the deacetylase gene
  • the plasmids plB17 1 and plB17 2 were therefore transferred to the Rhizobium meliloti strain 2011 by means of a two-factor crossing.
  • Incubation of R meliloti wild-type strains with radioactively labeled N-acetyl-PTC showed that this strain N-acetyl-PTC was not deacety ert (after incubation of plB17 1-carrying strains with N-acetyl-PTC and N-acetyl-PTT, deacetylation can be demonstrated by means of thin-layer chromatography). It was also possible to show that R meliloti is very sensitive to PTC and PTT. Therefore, deacetylation can be carried out also demonstrate the inhibition of R meliloti glutamine synthetases by the released PTC Example 3: Transfer of the modified deacetylase gene in Nicotiana tabacum
  • the deacetylase gene modified in Example 1 was transferred to A tumefaciens LBA4404 by means of a 2-factor crossing.
  • the resulting strains LBA4404 / 17 1 and LBA4404 / 17 2 were used to incubate Nicotiana tabacum leaf disks and, after 3 days, reacted to a sprout induction medium containing kanamycin. Regenerating kanamycin-resistant can be tested for the presence of the deacetylase gene by Southern hybridization. After treatment with N-acetyl-PTC or N-acetyl-PTT, the plants are then killed by the released PTC or PTT
  • Example 4 Construction of a vector for the transient expression of the modified deacetylase gene in E. coli and tobacco protoplasts
  • the modified deacetylase gene from plB17 1 and plB17 2 was excised from the plasmids by EcoRI / HindIII digestion. The restricted DNA was separated in an agarose gel and a 0.9 kb fragment was isolated in each case.
  • the vector pSVB28 (Arnold and Puhler, Gene 70, 171-179, 1988) has just f alls with EcoRI / HindIII digested the two batches were combined and ligated After transformation into the beta-galactosidase-negative E. coli strain JM83 showed all vector carrying clones a blue color, whereas clones which carry a vector with insertion of the deacetylase gene , remained white.
  • the plasmid DNA was isolated from the E. coli strains constructed in Example 4. Young tobacco leaves were incubated with digestive enzymes for 20 hours. The protoplasts falling from the skeleton were cleaned and incubated in a transfer buffer with polyethylene glycol (PEG) and the isolated DNA. The protoplasts were then washed and taken up in a culture liquid (K3 medium). After 3 days of incubation under dim lighting, the regenerating protoplasts were digested and the crude extracts were incubated with radioactively labeled N-acetyl-PTC and N-acetyl-PTT. The deacetylated PTC or PTT can be detected by thin layer chromatography.
  • PEG polyethylene glycol
  • Example 6 Method for producing male-sterile crop plants using the deacetylase gene from S. viridochromogenes under the control of a wallpaper-specific promoter.
  • the deacetylase gene from Streptomyces viridochromogenes is fused with a pistol-specific promoter and introduced into the tobacco cells by means of leaf disc transformation mediated by agrobacteria.
  • the plants regenerating from these cells are sprayed with N-acetyl-PTC or N-acetyl-PTT at any time before flowering. It can be shown that N-acetyl-PTC is stable in the plant row and is transported into all cells. Neither of the two substances has recognizable negative consequences for the wild type plant. As soon as the first pistil cells form, they begin to express the deacetylase gene.
  • the N-acetyl-PTC or N-acetyl-PTT stored in the cell is deacetylated by the enzyme and thus converted into its effective form. It inhibits the cells' glutamine synthetase and thus leads to rapid death. Functional embryos or seeds can no longer arise. Nevertheless, the development of the male reproductive system is impaired. In addition, the formation of deacetylase is interrupted. Surrounding cells are not impaired. If the plant is not treated with N-acetyl-PTC or N-acetyl-PTT, it is fully fertile. This eliminates the need to cancel the fs by a gene from the male partner of the cross. At the same time, a precisely defined mutation is present that is without Effects on the vigor and usability of the plant remains

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé permettant d'obtenir des plantes transgéniques en utilisant des promoteurs à spécificité tissulaire. Dans lesdites plantes, le développement de certaines parties peut être inhibé de façon ciblée.
EP97909268A 1996-09-26 1997-09-15 Procede de production de plantes a sterilite femelle Ceased EP0928338A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19639463 1996-09-26
DE19639463A DE19639463A1 (de) 1996-09-26 1996-09-26 Verfahren zur Herstellung weiblich steriler Pflanzen
PCT/EP1997/005037 WO1998013504A1 (fr) 1996-09-26 1997-09-15 Procede de production de plantes a sterilite femelle

Publications (1)

Publication Number Publication Date
EP0928338A1 true EP0928338A1 (fr) 1999-07-14

Family

ID=7806895

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97909268A Ceased EP0928338A1 (fr) 1996-09-26 1997-09-15 Procede de production de plantes a sterilite femelle

Country Status (9)

Country Link
US (2) US6759572B2 (fr)
EP (1) EP0928338A1 (fr)
JP (1) JP2001501088A (fr)
CN (1) CN1231699A (fr)
BR (1) BR9711552A (fr)
CA (1) CA2265938A1 (fr)
DE (1) DE19639463A1 (fr)
HU (1) HUP9904347A3 (fr)
WO (1) WO1998013504A1 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6555733B2 (en) 1996-12-16 2003-04-29 Hoechst Schering Agrevo Gmbh Genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and use
DE19652284A1 (de) 1996-12-16 1998-06-18 Hoechst Schering Agrevo Gmbh Neue Gene codierend für Aminosäure-Deacetylasen mit Spezifität für N-Acetyl-L-Phosphinothricin, ihre Isolierung und Verwendung
HUP0002390A3 (en) * 1997-03-03 2002-08-28 Syngenta Participations Ag Method of hybrid seed production using conditional female sterility
US6815577B1 (en) 1997-03-03 2004-11-09 Syngenta Participations Ag Method of hybrid seed production using conditional female sterility
EP0987331A1 (fr) * 1998-09-01 2000-03-22 Hoechst Schering AgrEvo GmbH Controlle de la pathogenicite des plantes a travers l'expression pathogène-inducible du gène deac
EP0987330A1 (fr) * 1998-09-01 2000-03-22 Hoechst Schering AgrEvo GmbH Modification du developpement et de la differentiation des plantes par moyen de l'expression tissue-specifique du gene Deac
US6384304B1 (en) 1999-10-15 2002-05-07 Plant Genetic Systems N.V. Conditional sterility in wheat
WO2002072804A2 (fr) * 2001-03-12 2002-09-19 Bayer Bioscience N.V. Nouveaux genes pour ablation cellulaire conditionnelle
US7152786B2 (en) * 2002-02-12 2006-12-26 Digimarc Corporation Identification document including embedded data
AU2003207323B2 (en) 2002-02-26 2007-09-13 Syngenta Limited A method of selectively producing male or female sterile plants
US20050044596A1 (en) * 2003-03-19 2005-02-24 Smith Alan G. Methods to confer enhanced floral properties to plants
CA2529614C (fr) * 2003-07-08 2012-05-15 Syngenta Limited Procede de production selective de plantes steriles males ou femelles

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK0412006T3 (da) * 1989-08-04 2001-03-05 Aventis Cropscience Nv Planter med modificerede blomster, frø eller embryoer
DE4126414A1 (de) 1991-08-09 1993-02-11 Hoechst Ag Deacetylasegene zur erzeugung von phosphinothricin oder phosphinothricyl-alanyl-alanin, verfahren zu ihrer isolierung und ihre verwendung
DE4308061A1 (de) * 1993-03-13 1994-09-15 Hoechst Ag Neue Aminosäure-Deacetylase mit Spezifität für L-N-Acetyl-Phosphinothricin, ihre Herstellung und Verwendung
WO1994025613A1 (fr) * 1993-05-03 1994-11-10 Cornell Research Foundation, Inc. Elements d'adn isoles dirigeant l'expression d'un gene specifique au pistil et a l'anthere, et leur procede d'utilisation
HUP0002390A3 (en) * 1997-03-03 2002-08-28 Syngenta Participations Ag Method of hybrid seed production using conditional female sterility

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9813504A1 *

Also Published As

Publication number Publication date
DE19639463A1 (de) 1998-04-02
AU4703397A (en) 1998-04-17
HUP9904347A3 (en) 2002-01-28
JP2001501088A (ja) 2001-01-30
US20020002710A1 (en) 2002-01-03
CA2265938A1 (fr) 1998-04-02
AU716327B2 (en) 2000-02-24
WO1998013504A1 (fr) 1998-04-02
US6759572B2 (en) 2004-07-06
US6852909B2 (en) 2005-02-08
HUP9904347A2 (hu) 2000-05-28
US20030051274A1 (en) 2003-03-13
CN1231699A (zh) 1999-10-13
BR9711552A (pt) 1999-08-24

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