EP1315801A1 - Hydroxy-phenyl pyruvate dioxygenase die zu einem peptidesignal fusioniert ist, dna sequenz und herbizidtolerante pflanzen, die ein solches gen enthalten - Google Patents

Hydroxy-phenyl pyruvate dioxygenase die zu einem peptidesignal fusioniert ist, dna sequenz und herbizidtolerante pflanzen, die ein solches gen enthalten

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Publication number
EP1315801A1
EP1315801A1 EP00962580A EP00962580A EP1315801A1 EP 1315801 A1 EP1315801 A1 EP 1315801A1 EP 00962580 A EP00962580 A EP 00962580A EP 00962580 A EP00962580 A EP 00962580A EP 1315801 A1 EP1315801 A1 EP 1315801A1
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EP
European Patent Office
Prior art keywords
hppd
chimeric gene
plants
fusion protein
coding
Prior art date
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EP00962580A
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English (en)
French (fr)
Inventor
Jean-Marc Ferullo
Eric Paget
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Bayer CropScience SA
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Bayer CropScience SA
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Publication date
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Publication of EP1315801A1 publication Critical patent/EP1315801A1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the present invention relates to a nucleic acid sequence coding for a hydroxy-phenyl pyruvate dioxygenase (HPPD) fused to a signal peptide, a chimeric gene containing this sequence as coding sequence and its use for obtaining plants resistant to certain herbicides.
  • HPPD hydroxy-phenyl pyruvate dioxygenase
  • Hydroxy-phenyl pyruvate dioxygenases are enzymes that catalyze the reaction to convert para-hydroxy-phenyl-pyruvate (HPP) to homogentisate. This reaction takes place in the presence of iron (Fe 2+ ) in the presence of oxygen (Crouch NP & al., Tetrahedron, 53, 20, 6993-7010, 1997).
  • Such herbicides targeting HPPD described in the prior art are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276 ) in particular isoxaflutole, a selective corn herbicide, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-1- (2-S ⁇ 2 CH3-4-CF3 phenyl) propane- 1,3-dione and 2-cyano-3-cyclopropyl-1- (2-S ⁇ 2 CH3-4-2,3 CI2 phenyl) propane-1,3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione or mesotrione, or also pyrazolinates.
  • isoxazoles EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482,
  • HPPDs are enzymes with cytoplasmic localization (Garcia, I., et al. 1997. Biochem. J. 325: 761-769 ..), it has also been shown that better tolerance was obtained when exogenous HPPD was accumulated in plastids, more particularly chloroplasts.
  • the present invention therefore relates to a fusion protein constituted by an HPPD fused at its C-terminal or N-terminal end to a signal protein sequence allowing the addressing of HPPD in cellular compartments other than the cytoplasm or the plastids (in especially chloroplasts).
  • HPPD is meant according to the invention any native HPPD enzyme, mutated or chimeric, exhibiting HPPD activity.
  • Many HPPDs are described in the literature, in particular the HPPDs of bacteria such as Pseudomonas (R ⁇ etschi & al., Eur. J. Biochem., 205, 459-466, 1992, WO 96/38567), of plants such as Arabidopsis ( WO 96/38567, Genebank AF047834) or carrot (WO 96/38567, Genebank 87257), Coccicoides (Genebank COITRP), or mammals such as mice or pigs.
  • Pseudomonas Roseudomonas
  • Arabidopsis WO 96/38567, Genebank AF047834
  • carrot WO 96/38567, Genebank 87257
  • Coccicoides Genebank COITRP
  • mutated HPPD is understood to mean, according to the invention, mutated HPPDs in order to obtain improved properties of tolerance to herbicides inhibiting HPPD compared to the corresponding native HPPD.
  • the mutated HPPD is a mutated HPPD in its C-terminal part as described in patent application WO 99/24585.
  • chimeric HPPD is meant a HPPD comprising elements originating from different HPPDs, in particular the chimeric HPPDs described in patent application WO 99/24586.
  • the HPPD is an HPPD from Pseudomonas fluorescens (WO
  • the mutated HPPD comprises the mutation W336 as described in patent application WO 99/24585.
  • signal protein sequence is meant according to the invention any signal or transit peptide allowing the addressing of HPPD in cellular compartments other than the cytoplasm or the plasts (in particular chloroplasts).
  • the signal protein sequence allows the addressing of the HPPD towards the vacuolar compartment (vacuole).
  • Such peptides are widely described in the literature (Neuhaus J.M. and Rogers J.C Sorting of proteins to vacuoles in plant cells Plant molecular Biology 38: 127-144, 1998).
  • the fusion protein is described by the sequence identifier No. 3 or 4 (SEQ ID NO 3 or 4) with its coding sequence.
  • the present invention also relates to a nucleic acid sequence coding for the fusion protein according to the invention.
  • the present invention also relates to the sequences capable of selectively hybridizing with the above nucleic acid sequence, the sequences homologous to the above sequence, the fragments of said sequences, and the sequences which differ from the sequences herein. above but which due to the degeneracy of the genetic code, code for the same fusion protein.
  • nucleic acid sequence means a nucleotide or polynucleotide sequence, which may be of DNA or RNA type, preferably of DNA type, in particular double strand.
  • sequence capable of hybridizing selectively is meant according to the invention the sequences which hybridize with the above sequences at a level above the background noise significantly.
  • the background noise can be linked to the hybridization of other DNA sequences present, in particular other cDNAs present in a cDNA library.
  • the level of the signal generated by the interaction between the sequence capable of hybridizing selectively and the sequences defined by the above sequence ID according to the invention is generally 10 times, preferably 100 times more intense than that of l interaction of other DNA sequences generating background noise.
  • the level of interaction can be measured, for example, by labeling the probe with radioactive elements, such as 32 P.
  • Hybridization is generally obtained by using very harsh environmental conditions (for example 0.03 M NaCl and 0.03 M sodium citrate at about 50 ° C-60 ° C). Hybridization can of course be carried out according to the usual methods of the prior art (in particular Sambrook & al, 1989, Molecular Cloning: A Labratory Manual).
  • homologous a nucleic acid fragment having one or more sequence modifications with respect to the nucleotide sequence coding for the fusion protein according to the invention. These modifications can be obtained according to the usual mutation techniques, or alternatively by choosing the synthetic oligonucleotides used in the preparation of said sequence by hybridization. In view of the multiple combinations of nucleic acids which can lead to the expression of the same amino acid, the differences between the reference sequence according to the invention and the corresponding homolog can be significant.
  • the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%.
  • These modifications are generally and preferably neutral, i.e. they do not affect the primary sequence of the fusion protein. This definition also applies to the protein sequence of the fusion protein, the mutations in this case being at the amino acid level and the degrees of homology being defined with respect to the primary sequence of the fusion protein.
  • the methods for measuring and identifying the homologies between the nucleic acid sequences are well known to those skilled in the art.
  • the methods for measuring and identifying homologies between polypeptides or proteins are also known to those skilled in the art.
  • fragments is meant according to the invention fragments of the DNA sequences according to the invention, that is to say the above sequences for which parts have been deleted but which retain the function of said sequences, c 'ie the addressing function for the small signal and the HPPD activity for the protein HPPD.
  • nucleic acid sequence coding for the fusion protein according to the invention in a process for the transformation of plants, as a marker gene or as a coding sequence making it possible to confer on the plant. tolerance to herbicides that inhibit HPPD.
  • This sequence can of course also be used in combination with other marker gene (s) and / or coding sequence (s) for one or more agronomic properties.
  • the present invention also relates to a chimeric gene (or expression cassette) comprising a coding sequence as well as heterologous 5 ′ and 3 ′ regulatory elements capable of functioning in a host organism, in particular plant cells or plants, the coding sequence comprising at least one nucleic acid sequence coding for a fusion protein as defined above.
  • host organism any mono or multicellular organism, lower or higher, into which the chimeric gene according to the invention can be introduced, for the production of mutated HPPD.
  • bacteria for example E. coli, yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, of a baculovirus, or preferably plant cells and plants.
  • plant cell any cell originating from a plant and which can constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds.
  • plant means any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food, such as corn, wheat, rapeseed, soy, rice, sugarcane, beet, tobacco, cotton, etc.
  • the regulatory elements necessary for the expression of the nucleic acid sequence coding for a fusion protein according to the invention are well known to those skilled in the art depending on the host organism. They include in particular promoter sequences, transcription activators, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to those skilled in the art and widely described in the literature.
  • the invention relates more particularly to the transformation of plants.
  • Promoters As promoter regulatory sequence in plants, use may be made of any promoter sequence of a gene expressing itself naturally in plants, in particular a promoter expressing in particular in the leaves of plants, such as promoters known as constitutive of bacterial, viral or plant origin or so-called light-dependent promoters such as that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit of a plant or any suitable known promoter which can be used.
  • promoters of plant origin mention will be made of the histone promoters as described in application EP 0 507 698, or the rice actin promoter (US Pat. No. 5,641,876).
  • the promoters of a plant virus gene there may be mentioned that of the cauliflower mosaic (CAMN 19S or 35 S), or the circovirus promoter (AU 689 311).
  • the promoter is chosen from light-dependent promoters (WO 99/25842), the rice actin promoter, the histone promoters and the promoters constituted by the fusion of a histone promoter and the first intron of rice actin (WO 99/34005).
  • WO 99/25842 light-dependent promoters
  • the rice actin promoter the histone promoters
  • transcription activators such as transcription activators (enhancer”
  • NMT tobacco mosaic virus
  • TEN tobacco etch virus
  • any corresponding sequence of bacterial origin such as for example the terminator nos of Agrobacterium tumefaciens, of viral origin, such as for example the terminator of CaMN 35 S, or of vegetable origin, such as for example a histone terminator as described in application EP 0 633 317.
  • the present invention also relates to a cloning and / or expression vector for the transformation of a host organism containing at least one chimeric gene as defined above.
  • This vector comprises, in addition to the above chimeric gene, at least one origin of replication.
  • This vector can consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the chimeric gene according to the invention.
  • transformation vectors as a function of the host organism to be transformed are well known to those skilled in the art and widely described in the literature.
  • the vector for transforming plant cells or plants according to the invention is a plasmid.
  • the vector comprises, in addition to the chimeric gene according to the invention, at least one second chimeric gene functional in the same host organism, comprising a nucleic acid sequence coding for an HPPD for addressing in a cell compartment other than the cell compartment in which the HPPD of the first chimeric gene is addressed.
  • the second chimeric gene can be another chimeric gene according to the invention allowing the addressing of HPPD in the lumen of the thylakoid or the thylakoid membrane, in the endoplasmic reticulum, the cell wall or the mitochondria, or a chimeric gene as described in the prior art allowing the addressing of HPPD either in the cytoplasm or in the chloroplasts .
  • the second chimeric gene allows the addressing of HPPD in the chloroplasts. It comprises, in addition to the regulatory elements defined above, allowing expression of the coding sequence in a host organism, a nucleic acid sequence coding for a transit peptide / HPPD fusion protein.
  • the transit peptide makes it possible to address the chimeric HPPD in the plastids, more particularly the chloroplasts, the fusion protein being cleaved between the transit peptide and the chimeric HPPD in the passage of the plastid membrane.
  • the transit peptide can be simple, like an EPSPS transit peptide (described in US Pat. No.
  • the vector comprises, in addition to the chimeric gene according to the invention, and where appropriate a second chimeric gene coding for an HPPD, another chimeric gene coding for another gene of interest.
  • a second chimeric gene coding for an HPPD another chimeric gene coding for another gene of interest.
  • It may be a gene coding for a selection marker as well as a gene conferring on the transformed plant new agronomic properties, or a gene for improving agronomic quality of the transformed plant.
  • Selection Markers Among the genes coding for selection markers, there may be mentioned antibiotic resistance genes, herbicide tolerance genes (bialaphos, glyphosate or isoxazoles), genes coding for easily identifiable reporter enzymes such as the enzyme GUS, genes coding for pigments or enzymes regulating the production of pigments in transformed cells.
  • selection marker genes are described in particular in patent applications EP 242 236, EP 242 246, GB 2 197 653, WO 91/02071, WO 95/06128, WO
  • genes conferring new agronomic properties on transformed plants there may be mentioned the genes conferring tolerance to certain herbicides, those conferring resistance to certain insects, those conferring tolerance to certain diseases, etc. Such genes are described in particular in patent applications WO 91/02071 and WO 95/06128.
  • Herbicide tolerance Among the genes conferring tolerance to certain herbicides, mention may be made of the Bar gene conferring tolerance to bialaphos, the gene coding for an appropriate EPSPS conferring resistance to herbicides having EPSPS as target such as glyphosate and its salts (US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 5,633,435, FR 2,736,926), the gene coding for glyphosate 5,4 oxidized.
  • glyphosate and its salts US 4,535,060, US 4,769,061, US 5,094,945, US 4,940,835, US 5,188,642, US 4,971,908, US 5,145,783, US 5,310,667, US 5,312,910, US 5,627,061, US 5,633,435, FR 2,
  • EPSPS EPSPS
  • FR 2 736 926 hereinafter referred to as double mutant EPSPS
  • CP4 the gene coding for an EPSPS isolated from Agrobacterium described by the sequences ID 2 and ID 3 of US patent 5,633,435, hereinafter referred to as CP4.
  • sequence coding for these enzymes is advantageously preceded by a sequence coding for a transit peptide, in particular for the transit peptide as defined above. above.
  • Resistance to Insects Among the proteins of interest conferring new properties of resistance to insects, mention will be made more particularly of the Bt proteins widely described in the literature and well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorabdus (WO 97/17432 & WO 98/08932).
  • chitinases glucanases, oxalate oxidase, all these proteins and their coding sequences being widely described in the literature, or else antibacterial and / or antifungal peptides, in particular peptides of less than 100 amino acids rich in cysteines such as thionines or plant defensins, and more particularly lyric peptides of all origins comprising one or more disulfide bridges between cysteines and regions comprising basic amino acids, in particular the following lytic peptides: androctonine (WO 97/30082 and WO 99/09189), drosomicine (WO 99/02717) or thanatine (WO 99/24594).
  • cysteines such as thionines or plant defensins
  • lyric peptides of all origins comprising one or more disulfide bridges between cysteines and regions comprising basic amino acids in particular the following lytic peptides: androctonine (WO 97/
  • the protein or peptide of interest is chosen from fungal eliciting peptides, in particular elicitins (Kamoun & al, 1993; Panab restaurants & al, 1995). Change in quality
  • proteins enriched in sulfur amino acids will also have the function of trapping and storing excess cysteine and / or methionine, making it possible to avoid the possible problems of toxicity linked to an overproduction of these sulfur amino acids by trapping them.
  • Mention may also be made of genes coding for peptides rich in sulfur amino acids and more particularly in cysteines, the said peptides also having antibacterial and / or antifungal activity.
  • Mention will be made more particularly of plant defensins, as well as lytic peptides of any origin, and more particularly the following lytic peptides: androctonine, drosomicine or thanatine.
  • the subject of the invention is also a method of transforming host organisms, in particular plant cells by integration of at least one nucleic acid sequence or a chimeric gene as defined above, a transformation which can be obtained by any appropriate known means, fully described in the specialized literature and in particular the references cited in the present application, more particularly by the vector according to the invention.
  • a series of methods involves bombarding cells, protoplasts or tissues with particles to which the DNA sequences are attached.
  • Another series of methods consists in using as a means of transfer into the plant a chimeric gene inserted into a Ti plasmid of Agrobacterium tumefaciens or Ri of Agrobacterium rhizogenes.
  • Other methods can be used such as micro-injection or electroporation, or even direct precipitation using PEG.
  • Those skilled in the art will choose the appropriate method depending on the nature of the host organism, in particular the plant cell or the plant.
  • Another subject of the present invention is host organisms, in particular plant cells or plants, transformed and containing a chimeric gene comprising a sequence coding for a fusion protein defined above.
  • the chimeric gene is stably integrated into the genome of the host organism.
  • the host organism comprises, in addition to the chimeric gene according to the invention, at least one second chimeric gene functional in the same host organism, comprising a sequence of nucleic acid coding for an HPPD for addressing in a cell compartment other than the cell compartment in which the HPPD of the first chimeric gene is addressed as defined above.
  • the host organism comprises, in addition to the chimeric gene according to the invention, and where appropriate a second chimeric gene coding for an HPPD, another gene chimera coding for another gene of interest as defined above.
  • the various chimeric genes can be integrated into the genome of the host organism by transformation using a vector according to the invention comprising the various chimeric genes as defined above.
  • the various chimeric genes can also be stably integrated into the genome of the host organism by co-transformation by means of several vectors, each comprising at least one chimeric gene which must be integrated into the genome of the host organism.
  • Another subject of the present invention is transformed plants, in the genome of which at least one chimeric gene according to the invention is stably integrated.
  • the plants according to the invention contain transformed cells as defined above, in particular plants regenerated from the transformed cells above.
  • the regeneration is obtained by any suitable process which depends on the nature of the species, as for example described in the references above.
  • the present invention also relates to the transformed plants resulting from the culture and / or the crossing of the regenerated plants above, as well as the seeds of transformed plants.
  • the transformed plants comprise, in addition to the chimeric gene according to the invention, at least one second chimeric gene functional in the same host organism, comprising a nucleic acid sequence coding for an HPPD for addressing in a cell compartment other than the cell compartment in which the HPPD of the first chimeric gene is addressed as defined above.
  • the transformed plants comprise, in addition to the chimeric gene according to the invention, and where appropriate a second chimeric gene coding for an HPPD, another chimeric gene coding for another gene of interest such as defined above.
  • the various chimeric genes in plants transformed according to the invention can come either from the same parent transformed plant, and in this case the plant comes from a single transformation / regeneration process with the different chimeric genes contained in the same vector or by co-transformation using several vectors. It can also be obtained by crossing parent plants each containing at least one chimeric gene, one of the parent plants comprising at least one chimeric gene according to the invention.
  • the transformed plants obtainable according to the invention can be of the monocotyledon type such as for example cereals, sugar cane, rice and corn or dicotyledons such as for example tobacco, soybeans, rapeseed, cotton, beet, clover, etc.
  • the subject of the invention is also a process for selective weeding of plants, in particular of crops, using an HPPD inhibitor, in particular a herbicide defined previously, characterized in that this herbicide is applied to transformed plants. according to the invention, both in pre-plant, pre-emergence and post-emergence of the culture.
  • the present invention also relates to a method of controlling weeds in a surface of a field comprising seeds or plants transformed with the chimeric gene according to the invention, which method consists in applying in said field surface a toxic dose for the said weeds of a herbicide inhibiting HPPD, without however substantially affecting the seeds or plants transformed with the said chimeric gene according to the invention.
  • the present invention also relates to a process for cultivating plants transformed according to the invention with a chimeric gene according to the invention, which process consists in sowing the seeds of said transformed plants in a surface of a field suitable for the cultivation of said plants, to apply to said surface of said field a dose toxic to weeds of a herbicide targeting HPPD defined above in the presence of weeds, without substantially affecting said seeds or said plants then harvest the cultivated plants when they reach the desired maturity and possibly separate the seeds from the harvested plants.
  • the application of the herbicide targeting HPPD can be carried out according to the invention, both in pre-planting, pre-emergence and post-emergence of the crop.
  • herbicide within the meaning of the present invention is meant a herbicidal active material alone or associated with an additive which modifies its effectiveness such as for example an agent increasing activity (synergist) or limiting activity (in English safener).
  • an agent increasing activity synergist
  • limiting activity in English safener
  • HPPD inhibitor herbicides are defined above.
  • the above herbicides are associated in known manner with the adjuvants of formulations usually used in agrochemistry
  • the method according to the invention may comprise the simultaneous or delayed application of a HPPD inhibitor in combination with said herbicide, for example glyphosate.
  • Another subject of the invention is the use of the chimeric gene coding for a fusion protein according to the invention as a marker gene during the "transformation-regeneration" cycle of a plant species and selection on the above herbicide.
  • the chimeric gene according to the invention When the chimeric gene according to the invention is combined in the same vector with another chimeric gene coding for a protein of interest conferring new agronomic properties other than a herbicide tolerance gene (resistance to insects, resistance to diseases, modification of the quality), the application of the herbicide inhibitor of HPPD on the transformed plants and their descendants, makes it possible to select in the fields the plants resulting from a cross between a parent plant comprising the two chimeric genes and an unprocessed plant who will have preserved the chimeric genes.
  • a herbicide tolerance gene resistance to insects, resistance to diseases, modification of the quality
  • Clone O a derivative of pRP O described in patent WO 96/38567 containing an expression cassette for HPPD, double histone promoter - TEV - HPPD gene - nos terminator.
  • pRP O was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I described in P. Hajdukiewicz et al Plant Molecular Biology 25: 989 -994, 1994.
  • Clone Q a derivative of pRP Q described in patent WO 96/38567 containing an expression cassette for HPPD, double histone promoter - TEV - OTP - HPPD gene - nos terminator.
  • pRP Q was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I, pPZP 212.
  • Vacuole clone the vacuole peptide used in the context of this work is that described in the following article: J.M. FeruUo et al Plant Molecular Biology 33: 625-633, 1997
  • Transit peptide vacuole This peptide was produced using two successive PCRs.
  • the first is carried out with the aim of synthesizing the vacuole transit peptide.
  • the oligonucleotides chosen have the following sequence:
  • VAC 1 5 'AAGATCAAGG ACAAAATTCA TGGTGAAGGT GCAGATGGTG AAAAGAAAAA
  • GAAGAAGGAG AAGAAGAAAC ATG 3 'VAC 2 5' ATCACTGTCA CTGCTGCTGC TATCATGGCC ATGTTCATGA CCCTCTCCAT
  • GTTTCTTCTT CTCCTTCTTC 3 These oligonucleotides hybridize to each other in the region shown in bold and thus make it possible to reconstitute the vacuole transit peptide which comprises 123 base pairs.
  • the reaction was carried out in a HYBAID PCR device with Taq polymerase PERKIN ELMER in its buffer under standard conditions, that is to say for 50 ⁇ l of reaction the dNTPs are mixed with 200 ⁇ M, the Taq polymerase with 2.5 units and 0 , 5 ⁇ l of each oligonucleotide at 10 ⁇ M.
  • a single PCR cycle was carried out, it consists of 5 min at 95 ° C then 1 min at 64 ° C then 10 min at 72 ° C.
  • the second PCR was used to add the Xcm I and Sal I restriction sites necessary for cloning into pRP O.
  • the oligonucleotides used have the following sequences: VAC 3: 5 'ACGTCCAGGT GCGTCGTGGT GTATTGACCG CCGATAAGAT CAAGGACAAA ATTC 3' VAC 4: 5 'ACGTGTCGAC TTAATCACTG TCACTGCTGC TG 3'
  • oligonucleotides make it possible to obtain an amplified fragment of 169 base pairs.
  • the reaction was carried out in a HYBAID PCR device with Taq polymerase PERKIN ELMER in its buffer under standard conditions, that is to say for 50 ⁇ l of reaction the dNTPs are mixed with 200 ⁇ M, the Taq polymerase with 2.5 units, 0 , 5 ⁇ l of each oligonucleotide at 10 ⁇ M and 1 ⁇ l of the first PCR.
  • the amplification program used is, 4 min at 95 ° C then 35 cycles ⁇ 30 sec 95 ° C, 30 sec 66 ° C, 1 min 72 ° C> followed by 10 min at 72 ° C.
  • n pRP O - vacuole a derivative of pRP O containing an HPPD expression cassette, double histone promoter - TEV - HPPD gene - vacuole transit peptide - terminator nos.
  • the plasmid pRP O was digested with Xcm I and Sal I and then ligated to the vacuole transit peptide previously purified and digested with Xcm I and Sal I.
  • n - vacuole clone a derivative of pRP O - vacuole containing a cassette d expression of HPPD, double histone promoter - TEV - HPPD gene - vacuole transit peptide - terminator nos.
  • pRP O - vacuole was digested with Sac I and Pvu II, the chimeric gene was purified and then ligated into pPZP 212 previously digested with Sac I and Sma I.
  • the vector is introduced into the non-oncogenic strain of Agrobacterium tumefaciens EHA 105.
  • Regeneration The regeneration of "Petit Havana" tobacco from leaf explants is carried out on a Murashig and Skoog (MS) base medium comprising 30 g / l of sucrose as well as 350 mg / I of cefotaxime and 200 mg / ml of kanamycin.
  • Leaf explants are removed on plants in the greenhouse and transformed using the leaf disc technique (Science 1985, Vol 227, pl229-1231) in successive stages:
  • the first includes the induction of shoots on an MS medium supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 15 days and 200 mg / ml of kanamycin.
  • MS medium supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 15 days and 200 mg / ml of kanamycin.
  • ANA naphthylacetic acid
  • BAP benzylaminopurine
  • the green shoots formed during this stage are then developed by culture on an MS medium supplemented with 30g / l of sucrose and 200 mg / ml of kanamycin, but containing no hormone, for 10 days.
  • the tolerance of transformed plants is studied by sowing on soil treated with isoxaflutole.
  • the first includes the induction of shoots on an MS medium supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 19 days and kanamycin + variable doses herbicide.
  • MS medium supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 19 days and kanamycin + variable doses herbicide.
  • ANA naphthylacetic acid
  • BAP benzylaminopurine
  • the second corresponds to transplanting the leaf discs (on which calluses and shoots have formed) on an MS medium supplemented with 30g / l of sucrose containing 0.01mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 3 days and kanamycin + variable doses of herbicide; tolerance measurement is performed at this time.
  • MS medium supplemented with 30g / l of sucrose containing 0.01mg / l of naphthylacetic acid (ANA) and 2mg / l of benzylaminopurine (BAP) for 3 days and kanamycin + variable doses of herbicide; tolerance measurement is performed at this time.
  • ANA naphthylacetic acid
  • BAP benzylaminopurine
  • shoots appear this means that they have integrated the kanamycin tolerance gene; if they have integrated the kanamycin tolerance gene, this also means that they have integrated the gene allowing the expression of HPPD from Pseudomonas fluorescens and its final localization in the cytoplasm, or the chloroplast or even in the vacuole. Indeed these two genes or constructs are linked on T-DNA. If the shoots are white this means that they come from a cell which has integrated the kanamycin tolerance gene (otherwise the sprout would not appear) and the HPPD tolerance gene but that is not sufficient for induce tolerance to the herbicide. The percentage of green seedlings compared to the number of seedlings (white and green) is therefore a measure of the tolerance induced by the gene.

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EP00962580A 2000-09-08 2000-09-08 Hydroxy-phenyl pyruvate dioxygenase die zu einem peptidesignal fusioniert ist, dna sequenz und herbizidtolerante pflanzen, die ein solches gen enthalten Withdrawn EP1315801A1 (de)

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FR2848568B1 (fr) * 2002-12-17 2005-04-15 Rhobio Gene chimere permettant l'expression d'une hydroxy-phenyl pyruvate dioxygenase dans les plastes et plantes transplastomiques contenant un tel gene tolerantes aux herbicides
CN103237894A (zh) * 2010-08-13 2013-08-07 先锋国际良种公司 包含具有羟基苯丙酮酸双加氧酶(hppd)活性的序列的组合物和方法

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FR2673643B1 (fr) * 1991-03-05 1993-05-21 Rhone Poulenc Agrochimie Peptide de transit pour l'insertion d'un gene etranger dans un gene vegetal et plantes transformees en utilisant ce peptide.
FR2734842B1 (fr) * 1995-06-02 1998-02-27 Rhone Poulenc Agrochimie Sequence adn d'un gene de l'hydroxy-phenyl pyruvate dioxygenase et obtention de plantes contenant un gene de l'hydroxy-phenyl pyruvate dioxygenase, tolerantes a certains herbicides
FR2751347B1 (fr) * 1996-07-16 2001-12-07 Rhone Poulenc Agrochimie Gene chimere a plusieurs genes de tolerance herbicide, cellule vegetale et plante tolerantes a plusieurs herbicides
FR2770854B1 (fr) * 1997-11-07 2001-11-30 Rhone Poulenc Agrochimie Sequence adn d'un gene de l'hydroxy-phenyl pyruvate dioxygenase et obtention de plantes contenant un tel gene, tolerantes aux herbicides
FR2778527A1 (fr) * 1998-05-18 1999-11-19 Rhone Poulenc Agrochimie Nouvelle methode de production de tocopherols dans les plantes et plantes obtenues

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