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
  • C12N15/8271—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
  • C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—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 biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43522—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from scorpions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a DNA sequence coding for androctonine, a vector containing it for the transformation of a host organism and the method of transformation of said organism.
• the invention relates more particularly to the transformation of plant cells and plants, the androctonine produced by the transformed plants giving them resistance to diseases, in particular of fungal origin.
• bactericidal or fungicidal properties in particular against the fungi responsible for plant diseases.
• the problem consists in finding such substances which can not only be produced by transformed plants, but also conserve their bactericidal or fungicidal properties and confer them on said plants.
• bactericide or fungicide means both the bactericidal or fungicidal properties proper as well as the bacteriostatic or fungistatic properties.
• the androctonines are peptides produced by scorpions, especially of the species Androctonus australis. An androctonine and its preparation by chemical synthesis is described by Ehret-Sabatier & coll., As well as its antifungal and antibacterial properties in vitro.
• genes androctonines could be inserted into a host organism, especially a plant to express a androctonin as for the preparation and isolation of this androctonin as to give the said host organism with properties of resistance to fungal diseases and diseases of bacterial origin, providing a particularly advantageous solution to problem stated above.
• the subject of the invention is therefore firstly a nucleic acid fragment coding for an androctonine, a chimeric gene comprising said fragment coding for an androctonine as well as regulatory elements in the heterologous 5 ′ and 3 ′ position which can function in an organism host, in particular in plants and a vector for the transformation of host organisms containing this chimeric gene, and the transformed host organism. It also relates to a transformed plant cell containing at least one nucleic acid fragment coding for an androctonine and a plant resistant to diseases containing the said cell, in particular regenerated from this cell. Finally, it relates to a process for growing transformed plants according to the invention.
• Xaa represents a peptide residue comprising at least 1 amino acid
• Xab represents a peptide residue of 5 amino acids
• Xac represents a peptide residue of 5 amino acids
• Xad represents a peptide residue of 3 amino acids
• Xae represents a peptide residue comprising at least 1 amino acid.
• Xab and / or Xad and / or Xae comprise at least one basic amino acid, preferably 1.
• basic amino acids is meant according to the invention the amino acids chosen from lysine, aspargine or homoaspargine.
• amino acids chosen from lysine, aspargine or homoaspargine Preferably,
• Xaa represents the peptide sequence Xaa '-Val, in which Xaa' represents NH2 or a peptide residue comprising at least 1 amino acid, and / or Xab represents the peptide sequence -Arg-Xab'-Ile, in which Xab 'represents a residue peptide of 3 amino acids, and / or
• Xac represents the peptide sequence -Arg-Xac'-Gly-, in which Xac 'represents a peptide residue of 3 amino acids, and / or Xad represents the peptide sequence -Tyr-Xad'-Lys, in which Xad * represents a peptide residue of 1 amino acid, and / or
• Xae represents the peptide sequence -Thr-Xae ', in which Xae " represents COOH or a peptide residue comprising at least 1 amino acid. More preferably,
• Xaa represents the peptide sequence Arg-Ser-, and / or
• Xab represents the peptide sequence -Gln-Ile-Lys-, and / or
• Xac represents the peptide sequence -Arg-Arg-Gly-, and / or
• Xad ' represents the peptide residue -Tyr-, and / or Xae' represents the peptide sequence -Asn-Arg-Pro-Tyr.
• androctonine is represented by the 25 amino acid peptide sequence described the sequence identifier No. 1 (SEQ ID NO 1) and the homologous peptide sequences.
• homologous peptide sequences any equivalent sequence comprising at least 65% homology with the sequence represented by the sequence identifier No. 1, it being understood that the 4 cysteine residues and the number of amino acids separating them remain identical , certain amino acids being replaced by different but equivalent amino acids on sites which do not induce a substantial modification of the antifungal or antibacterial activity of the said homologous sequence.
• the homologous sequences comprise at least 75% of homology, more preferably at least 85% of homology, even more preferably 90% of homology.
• the NH2-terminal residue of androctonine may have a post-translational modification, for example acetylation. whereas the C-terminal residue can present a post-translational modification. for example an amidation.
• peptide sequence essentially comprising the peptide sequence of general formula (I) is meant not only the sequences defined above, but also such sequences comprising at one or the other of their ends, or both, residues peptides necessary for their expression and targeting in a host organism, in particular a plant cell or a plant.
• the peptide fused to androctonine can be a signal peptide or a transit peptide which makes it possible to control and direct the production of androctonine in a specific way in a part of the host organism, in particular of the plant cell or of the plant, such as, for example, the cytoplasm, the cell membrane, or in the case of plants in a particular type of cell or tissue compartment or in the extracellular matrix.
• the transit peptide can be a chloroplastic or mitochondrial addressing signal, which is then cleaved in the chloroplasts or the mitochondria.
• the signal peptide can be an N-terminal signal or “prepeptide”, optionally in combination with a signal responsible for the retention of the protein in the endoplasmic reticulum, or an addressing peptide vacuolar or "propeptide".
• the endoplasmic reticulum is the place where the "cellular machinery" is responsible for the processing of the protein produced, such as for example the cleavage of the signal peptide.
• the transit peptides can be either single or double, and in this case optionally separated by an intermediate sequence, that is to say comprising, in the direction of transcription, a sequence coding for a transit peptide of a plant gene coding for a plastid localizing enzyme, part of the sequence of the mature N-terminal part of a plant gene coding for a plastid localizing enzyme, then a sequence coding for a second transit peptide of a plant gene coding for a enzyme with plastid localization, as described in application EP 0 508 909.
• transit peptide useful according to the invention include in particular the PR-l ⁇ tobacco gene signal peptide (WO 95/19443), represented with its coding sequence by the sequence identifier No. 2 (SEQ ID NO 2) and fused to androctonine by sequence identifier No. 3 (SEQ ID NO 3), in particular corresponding to the fusion protein corresponding to bases 12 to 176 of this sequence, in particular when androctonine is produced by plant or plant cells, or the precursor of the factor Mat l when androctonine is produced in yeasts.
• the present invention therefore firstly relates to a fragment of nucleic acid, in particular DNA, coding for l 'androctonine defined above.
• nucleic acid fragment is intended to mean a nucleotide sequence which may be of DNA or RNA type, preferably of DNA type. in particular cDNA, in particular double strand.
• the nucleic acid fragment encoding androctonin is the DNA sequence described by the sequence identifier No. 1 (SEQ ID NO 1), a homologous sequence or a sequence complementary to said sequence, more particularly the coding part of this SEQ ID NO1, corresponding to bases 1 to 75.
• homologous is meant according to the invention a fragment of nucleic acid having one or more sequence modifications relative to the nucleotide sequence described by the sequence identifier No. 1 and encoding androctonin. 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 may lead to the expression of a same amino acid, the differences between the reference sequence described by the sequence identifier No. 1 and the counterpart can be significant, especially since it is a question of a DNA fragment size of less than 100 nucleic acids, synthetically feasible.
• the degree of homology will be at least 70% compared to the reference sequence, preferably at least 80%, more preferably at least 90%. These modifications are generally neutral, i.e. they do not affect the primary sequence of the resulting androctonine.
• 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, linked operably linked to said coding sequence, said coding sequence comprising at least one DNA fragment encoding the androctonine as defined above (including the fusion peptide "androctonine peptide” or “androctonine-peptide”).
• host organism is meant any mono or multicellular organism. lower or higher, wherein the chimeric gene of the invention can be introduced, for the production of androctonine.
• These are in particular bacteria, for example E. coll. yeasts, in particular of the genera Saccharomyces or Kluyveromyces, Pichia, fungi, in particular Aspergillus, of a bacylovirus, 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 according to the invention 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 expression of the DNA fragment encoding the androctonin are well known to the skilled person depending on the host organism. They notably include promoter sequences, transcription activators, transit peptides, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to the skilled person.
• the regulatory elements are well known to those skilled in the art, and in particular include promoter sequences, trancription activators, transit peptides, terminator sequences and start codons. and stop.
• a DNA fragment coding for heliomycin is integrated into a shuttle vector which comprises the following elements:
• an expression cassette made up (1) a promoter regulation sequence
• yeasts of the species S. cerevisiae are transformed with the expression plasmid by the lithium acetate method (Ito et al., 1993, J. Bacteriol, 153, pp 163-168).
• the invention relates more particularly to the transformation of plants.
• 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 of bacterial, viral or plant origin such as, for example, that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit or a plant virus gene such as, for example, that of the cauliflower mosaic (CAMV 19S or 35S), or a promoter inducible by pathogens like tobacco PR-la, any known suitable promoter that can be used.
• a promoter of bacterial, viral or plant origin such as, for example, that of a gene for the small ribulose-biscarboxylase / oxygenase (RuBisCO) subunit or a plant virus gene such as, for example, that of the cauliflower mosaic (CAMV 19S or 35S), or a promoter inducible by pathogens like tobacco PR-la, any known suitable promoter that can be used.
• a promoter of bacterial, viral or plant origin such
• a promoter regulatory sequence which promotes the overexpression of the coding sequence in a constitutive manner or induced by the attack of a pathogen, such as for example, that comprising at least one histone promoter as described. in application EP 0 507 698.
• promoter regulatory sequence other regulatory sequences which are located between the promoter and the coding sequence, such as transcription activators (“enhancer”), such as for example the translational activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed.
• transcription activators such as for example the translational activator of the tobacco mosaic virus (TMV) described in application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed.
• any corresponding sequence of bacterial origin such as for example the terminator nos dAgrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in the application, can be used.
• the chimeric gene can also be associated with a selection marker adapted to the transformed host organism. Such selection markers are well known to those skilled in the art. It could be an antibiotic resistance gene, or a herbicide tolerance gene for plants.
• the present invention also relates to a cloning 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, and if appropriate an appropriate selection marker.
• 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 subject of the invention is also a method of transforming host organisms, in particular plant cells by integration of at least one nucleic acid fragment or a chimeric gene as defined above, a transformation which can be obtained by any appropriate known means widely described in the literature and in particular the references cited in this application, especially by the vector of 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 at Agrobacte ⁇ um tumefaciens or Ri of Agrobacterium rhizogenes.
• Other methods can be used such as micro-injection or electroporation, or even direct precipitation using PEG.
• the present invention also relates to host organisms, in particular plant cells or plants, transformed and containing an effective amount of a chimeric gene comprising a coding sequence for androctonine defined above.
• the present invention also relates to plants containing transformed cells, in particular plants regenerated from transformed cells.
• 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 plants thus transformed are resistant to certain diseases, in particular certain fungal or bacterial diseases. Therefore, the DNA sequence encoding androctonin can be integrated with the main objective for producing plants resistant to the said diseases, androctonine being effective against fungal diseases such as those caused by Cercospora, in particular Cercospora beticola ,
• Cladosporium in particular Cladosporium herbarum, Fusarium, in particular Fusarium culmorum or Fusarium gr amine arum, or by Phylophlhora, in particular Phytophtora cinnamomi.
• the chimeric gene may also be associated, and advantageously, with at least one selection marker, such as one or more herbicide tolerance genes.
• the DNA sequence coding for androctonine can also be integrated as a selection marker during the transformation of plants with other sequences coding for other peptides or proteins of interest, such as, for example, herbicide tolerance genes. .
• the cells and plants transformed according to the invention can comprise, in addition to the sequence coding for androctonine, other heterologous sequences coding for proteins of interest such as other complementary peptides capable of conferring on the plant resistance to other bacterial diseases or fungal, and / or other sequences coding for herbicide tolerance proteins, in particular defined above and / or other sequences coding for insect resistance proteins, such as Bt proteins in particular.
• the other sequences may be integrated by means of the same vector comprising the chimeric gene according to the invention, which comprises a sequence encoding the androctonine, and comprising at least another gene comprising another sequence encoding another peptide or protein of interest.
• Plants according to the invention can also be obtained by crossing parents, one carrying the gene according to the invention encoding the androctonine, the other carrying a gene encoding at least one other peptide or protein of interest.
• the present invention finally relates to a method for culturing transformed plants according to the invention, the method comprising pianter seeds of said transformed plants in an area of a culture medium, in particular a field appropriate for the cultivation of said plants, to apply an agrochemical composition to said surface, without substantially affecting said seeds or said transformed plants, then harvesting the cultivated plants when they reach the desired maturity and possibly separating the seeds from the harvested plants .
• agrochemical composition comprising at least one active product having one of the following activities, herbicidal, fungicidal, bactericidal, virucidal or insecticidal.
• the agrochemical composition comprises at least one active product having at least one fungicidal and / or bactericidal, more preferably exhibiting an activity complementary to that of produced by the androctonine transformed plants according to the invention.
• product having an activity complementary to that of androctonine is understood according to the invention a product having a spectrum of complementary activity, it is to say a product which will be active against attacks of contaminants (fungi, bacteria or viruses) insensitive to androctonin. or alternatively a product whose activity spectrum covers that of the androctonine. totally or in part, and whose application rate will be decreased substantially due to the presence of the androctonin produced by the transformed plant.
• the present invention therefore also relates to a process for preparing the androctonine, comprising the steps of culturing the transformed host organism comprising a gene encoding androctonin as defined above in a suitable culture medium, and then the total and partial extraction and purification of the androctonine obtained.
• pRPA-MD-P Creation of a plasmid containing the peptide signal of the tobacco PR-la gene.
• the two complementary synthetic oligonucleotides Oligo 1 and Oligo 2 below are hybridized at 65 ° C for 5 minutes and then by slowly decreasing the temperature to 30 ° C for 30 '.
• Oligo 1 5 'GCGTCGACGC GATGGGTTTC GTGCTTTTCT CTCAGCTTCC ATCTTTCCTT CTTGTGTCTA CTCTTCTTCT TTTCC 3'
• Oligo 2 5 'TCGCCGGCAC GGCAAGAGTA AGAGATCACA AGGAAAAGAA GAAGAGTAGA CACAAGAAGG AAAGATGGAA GC 3 • After hybridization between Oligo 1 and Oligo 2, the single stranded DNA serves as a template for the klenow fragment of E. coli polymerase 1 (under the standard conditions recommended by the manufacturer (New ⁇ ngland Biolabs)) for the creation of the double-stranded oligonucleotide from the 3 'end of each oligo.
• E. coli polymerase 1 under the standard conditions recommended by the manufacturer (New ⁇ ngland Biolabs)
• the double-stranded oligonucleotide obtained is then digested with the restriction enzymes Sac11 and Nael and cloned into the plasmid pBS II SK (-) (Stratagene) digested with the same restriction enzymes.
• a clone is then obtained comprising the region coding for signal peptide of the PR-1a gene from tobacco (S ⁇ Q ID NO 2).
• pRPA-PS-PRla-andro Creation of a sequence coding for androctonine fused to the signal peptide PR-la without region not transcribed in 3 '.
• Oligo 3 5 'AGGTCCGTGT GCAGGCAGAT CAAGATCTGC AGGAGGAGGG
• GTGG 3 'Oligo 4 5' CCGGATCCGT CGACACGTTC GCCTCGCCGA GCTCAGTATG
• a clone is then obtained comprising the region coding for the PR-la-androctonine fusion protein located between the Ncol restriction sites at the N-terminal end and Seal, Sacll and BamHI at the C-terminal end (S ⁇ Q ID NO 3).
• pRPA-RD-238 Creation of an expression vector in plants comprising the sequence coding for the fusion protein PR-la-androctonine.
• Plasmid pRTL-2 GUS derived from plasmid pUC-19, was obtained from Dr. Jim Carrington (Texas A&M University. Not described).
• This plasmid contains the duplicated CaMV 35S promoter isolated from the cauliflower mosaic virus (CaMV 2x35S promoter; Odell & al., 1985) which directs the expression of an RNA containing 5 'untranslated sequence of the tobacco etch virus (TEV 5'UTR; Carrington & Freed. 1990), the ⁇ -glucuronidase gene from E. coli (GUS Jefferson & coll., 1987) followed by the polyadenylation site of TARN 35S from CaMV (CaMV polyA; Odell & al., 1985).
• the plasmid pRTL-2 GUS is digested with the restriction enzymes Ncol and BamHI and the large DNA fragment is purified.
• the plasmid pRPA-PS-PRla-andro is digested with the restriction enzymes Ncol and BamHI and the small DNA fragment containing the region coding for the fusion protein PR-la-androctonine is purified.
• the two purified DNA fragments are then linked together in an expression cassette in plants which synthesizes a fusion protein PR-la-androctonine.
• the schematic structure of this expression cassette is represented in FIG. 2.
• "PR-la-androctonine" represents the coding region for the PR-la-androctonine fusion protein of pRPA-RD-230. Androctonine is transported to the extra-cellular matrix of the plant by the action of the signal peptide PR-la.
• pRPA-RD-195 Creation of a plasmid containing a modified multiple cloning site.
• Plasmid pRPA-RD-195 is a plasmid derived from pUC-19 which contains a modified multiple cloning site.
• the complementary synthetic oligonucleotides Oligo 5 and Oligo 6 below are hybridized and made double stranded according to the procedure described for pRPA-MD-P.
• Oligo 5 5 'AGGGCCCCCT AGGGTTTAAA CGGCCAGTCA GGCCGAATTC GAGCTCGGTA CCCGGGGATC CTCTAGAGTC GACCTGCAGG CATGC 3' Oligo 6: 5 'CCCTGAACCA GGCTCGAGGG CGCGCCTTAA TTAAAAGCTT GCATGACTCTC AGAG
• the double-stranded oligonucleotide obtained is then linked in pUC-19 which has been previously digested with the restriction enzymes EcoRI and HindIII and made ends up. Frank using the klenow fragment of DNA polymerase 1 from E. coli.
• a vector is obtained containing multiple cloning sites to facilitate the introduction of the expression cassettes into a vector plasmid grAgrobacterium tumefaciens. The schematic structure of this multiple cloning site is shown in Figure 3.
• pRPA-RD-233 Introduction of the PR-la-androctonine expression cassette from pRPA-RD-230 into pRPA-RD-195.
• the plasmid pRPA-RD-230 is digested with the restriction enzyme HindIII.
• the DNA fragment containing the PR-la-androctonine expression cassette is purified.
• the purified fragment is then linked in pRPA-RP-195 which was previously digested with the restriction enzyme HindIII and dephosphorylated with intestinal phosphatase from calves.
• pRPA-RD-174 Plasmid derived from pRPA-BL-150A ( ⁇ P 0 508 909) containing the bromoxynil tolerance gene from pRPA-BL-237 ( ⁇ P 0 508 909).
• the bromoxynil tolerance gene is isolated from pRPA-BL-237 by gene amplification by PCR. The fragment obtained is blunt-ended and is cloned into the EcoRI site of pRPA-BL-150A which has been blunt-blown by the action of the klenow polymerase under standard conditions.
• An Agrobacterium tumefaciens vector is obtained which contains the bromoxynil tolerance gene near its right border, a kanamycin tolerance gene near its left border and a multiple cloning site between these two genes.
• FIG. 4 The schematic structure of pRPA-RD-174 is shown in Figure 4.
• nos represents the polyadenylation site of nopaline synthase from Agrobacterium tumefaciens (Bevan & coll., 1983).
• NOS pro represents the promoter of the nopaline synthase of Agrobacterium tumefaciens (Bevan & coll., 1983),
• NPT II represents the gene for the neomycin phosphotransphérase of the transposon Tn5 of E.
• coli (Rothstein & coll., 1981)
• 35S pro represents the 35S promoter isolated from the cauliflower mosaic virus (Odell & al., 1985)
• BRX represents the nitrilase gene isolated from K. o ⁇ aenae (Stalker & al., 1988)
• RB and "LB” respectively represent the right and left borders of the sequence of a Ti plasmid of Agrobacterium tumefaciens.
• pRPA-RD-184 Addition of a new, unique restriction site in pRPA-RD-174.
• the complementary synthetic oligonucleotides Oligo 7 and Oligo 8 below are hybridized and made double stranded according to the procedure described for pRPA-MD-P.
• Oligo 7 5 'CCGGCCAGTC AGGCCACACT TAATTAAGTT TAAACGCGGC CCCGGCGCGC CTAGGTGTGT GCTCGAGGGC CCAACCTCAG TACCTGGTTC AGG 3'
• Oligo 8 5 'CCGGCCTGAA CCAGGTACTG AGGTTGGGCC CTCGAGCACA CACCTAGGCG CGCCGGGGCC GCGTTTAAAC TTAATTAAGT
• the hybrid double-stranded oligonucleotide (95 base pairs) is purified after separation on an agarose gel (3% Nusieve, FMC). Plasmid pRPA-RD-174 is digested with the restriction enzyme Xmal, and the large DNA fragment is purified. The two DNA fragments obtained are then linked.
• a plasmid derived from pRPA-RD-174 is obtained comprising other restriction sites between the bromoxynil tolerance gene and the selection marker kanamycin gene.
• the schematic structure of the plasmid pRPA-RD-184 is shown in Figure 5 where the terms "nos", “NPT II”. "NOS pro”, “35S pro”, “BRX gene”, “RB” and “LB” have the same meaning as in FIG. 4.
• pRPA-RD-236 Creation of a DAgrobacterium tumefaciens vector containing the construction of the gene coding for androctonine directed towards the extracellular matrix.
• the plasmid pRPA-RD-233 is digested with the restriction enzymes Pmel and Ascl and the DNA fragment containing the gene for PR-la-androctonine is purified.
• the plasmid pRPA-RD-184 is digested with the same restriction enzymes.
• the DNA fragment containing the PR-la-androctonine expression cassette is then linked in pRPA-RD-184. This gives an Agrobacterium tumefaciens vector containing the sequence coding for the PR-la-androctonine fusion protein which leads to the expression of androctonine in the extracellular matrix of the plant.
• Example 2 Tolerance to herbicides in processed tobacco.
• the vector pRPA-RD-236 is introduced into the Agrobacterium tumefaciens EHA101 strain (Hood & al., 1987) carrying the cosmid pTVK291 ( Komari & al., 1986).
• the transformation technique is based on the procedure of Horsh & coll. (1985).
• the regeneration of PBD6 tobacco (from SEITA France) from leaf explants is carried out on a Murashige and Skoog (MS) base medium comprising 30 g / l of sucrose as well as 200 ⁇ g / ml of kanamycin.
• the leaf explants are taken from plants cultivated in a greenhouse or in vitro and regenerated according to the technique of leaf discs (Horsh & coll., 1985) in three successive stages: the first comprises the induction of shoots on a medium supplemented with 30 g / 1 of sucrose containing 0.05 mg / 1 of naphthylacetic acid (ANA) and 2 mg / 1 of benzylaminopurine (BAP) for 15 days.
• ANA naphthylacetic acid
• BAP benzylaminopurine
• the shoots formed during this stage are then developed for 10 days by culture on an MS medium supplemented with 30 g / l of sucrose but containing no hormone. Then take developed shoots and cultivate them on an MS rooting medium with half content of salts, vitamins and sugar and containing no hormone. After about 15 days, the rooted shoots are put in the ground.
• MS medium supplemented with 30 g / l of sucrose but containing no hormone. The shoots formed during this stage are then developed for 10 days by culture on an MS medium supplemented with 30 g / l of sucrose but containing no hormone. Then take developed shoots and cultivate them on an MS rooting medium with half content of salts, vitamins and sugar and containing no hormone. After about 15 days, the rooted shoots are put in the ground.
• an MS medium supplemented with 30 g / l of sucrose but containing no hormone.
• MS rooting medium with half content of salts, vitamins and sugar and containing no hormone. After about 15 days, the rooted shoot

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Citations (5)

• 1997
  • 1997-08-20 FR FR9710632A patent/FR2767537B1/fr not_active Expired - Fee Related
• 1998
  • 1998-08-18 EP EP98942749A patent/EP1007711A1/fr not_active Withdrawn
  • 1998-08-18 JP JP2000509852A patent/JP2001514898A/ja active Pending
  • 1998-08-18 IL IL13460998A patent/IL134609A0/xx unknown
  • 1998-08-18 WO PCT/FR1998/001814 patent/WO1999009189A1/fr not_active Application Discontinuation
  • 1998-08-18 PL PL98338859A patent/PL338859A1/xx unknown
  • 1998-08-18 NZ NZ502951A patent/NZ502951A/en unknown
  • 1998-08-18 ZA ZA987450A patent/ZA987450B/xx unknown
  • 1998-08-18 AU AU90766/98A patent/AU751263B2/en not_active Ceased
  • 1998-08-18 CA CA002301978A patent/CA2301978A1/fr not_active Abandoned
  • 1998-08-18 HU HU0003585A patent/HUP0003585A3/hu unknown
  • 1998-08-19 HR HR9710632A patent/HRP980455A2/hr not_active Application Discontinuation
  • 1998-08-20 CO CO98047698A patent/CO4790113A1/es unknown
  • 1998-08-20 AR ARP980104113A patent/AR016839A1/es unknown

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