MXPA98000562A - 5-enol piruvilshikimato-3-phosphate mutated synase, gene that codifies for this protein, and transformed plants containing the - Google Patents

Abstract

The present invention describes a mutant glyphosate resistance gene of 5-enol pyruvyshikimate-3-phosphate synthase (EPSPS) which includes at least one substitution of threonine 102 for isoleucine, and useful for producing transformed plants resistant to glyphosa

Description

-ENOL PIRUV LSHIKIMATE-3-PHOSPHATE MUTATED SYNASE, GENE CODING FOR THIS PROTEIN, AND TRANSFORMED PLANTS CONTAINING THE GENE DESCRIPTION OF THE INVENTION The present invention relates to a new 5-enolpyruvylshikimate-3-phosphate synthase (or EPSPS) which exhibits an improved tolerance with respect to herbicides which are competitive inhibitors with respect to phosphoenolpyruvate (PEP) of EPSPS activity. This more tolerant EPSPS synthase possesses at least one substitution of "threonine for isoleucine". The invention also relates to a gene coding for such a protein, to cells of plants transformed by constructs of chimeric genes containing this gene, to plants regenerated from these cells, and also to plants that originate from cross using these transformed plants. Glyphosate, sulfosate and etine fossa are broad-spectrum systemic herbicides of the phosphonomethylglycine family. Act essentially as competitive inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) or EPSPS with respect to the PEP (phosphoenolpyruvate). After their application to the plant, they are translocated in the plant, where they accumulate in the rapidly growing parts, in particular the caulino and REF: 26706 apices of the roots, causing damage, to the point of destruction of sensitive plants. Plastid EPSPS, the main objective of these products, is an enzyme of the biosynthesis pathway of aromatic amino acids, which is encoded by one or more nuclear genes, and synthesized in the form of a cytoplasmic precursor, then imported into the plastid, where it accumulates in its mature form. The tolerance of plants to glyphosate and family products is obtained by the stable introduction into their genome of an EPSPS gene, of plant or bacterial origin, which is mutated or treated differently from the characteristics of inhibition by the glyphosate of the product of this gene. In view of the mode of action of glyphosate and the degree of tolerance to glyphosate of the product of the genes that are used, it is advantageous to be able to express the product of the translocation of this gene to allow it to accumulate in substantial amounts in the plastids. It is known, for example from US Pat. No. 4,535,060, to confer on a plant a tolerance to a herbicide of the above type, especially N-phosphonomethylglycine or glyphosate, by introducing into the plant genome a gene coding for an EPSPS that it carries at least one mutation that makes this enzyme more resistant to its competitive inhibitor (glyphosate) after the location of the enzyme in the plastid compartment. These techniques, however, need to be improved, to obtain greater reliability in the use of these plants under agricultural conditions. In the present description, "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, and "plant cell" is understood to mean any cell that originates from a plant, and is capable of constituting undifferentiated tissues such as calluses. , or differentiated tissues such as embryos or parts of plants or seeds. The subject of the present invention is the production of transformed plants having an improved tolerance to herbicides of the phosphonomethylglycine family, by regeneration of transformed cells by means of novel chimeric genes that contain a gene for tolerance to these herbicides. The subject of the invention is also a chimeric gene to confer on plants an increased tolerance with respect to a herbicide having the EPSPS as its target, comprising, in the direction of transcription: a promoter region, optionally a peptide region of transit, a sequence of a gene encoding an glyphosate-tolerance enzyme, and a non-translated polyadenylation signal region at the 3 'end, characterized in that the glyphosate tolerance gene contains, relative to the gene from which it is derived, a substitution of "threonine 102 for isoleucine" in the "aroA" region (EPSPS). Preferably, it also comprises, in the same region, a substitution of "proline 106 per serir.a". These substitutions can be introduced or be present in an EPSPS sequence of any origin, in particular of plant, bacterial, algal or fungal origin. Transit peptides that can be used in the region of the transit peptide can be, known per se, plant origin, for example those originating from the ai, sunflower, pea, tobacco or the like. The first and the second transit peptide can be identical, similar or different. They may, in addition, each comprise one or more transit peptide units according to European Patent Application EP 0 508 909. It is the role of this characteristic region to allow the release of a mature and natural protein, and especially the mutated EPSPS. from above, with maximum efficiency in the plasmid compartment.

The promoter region of the chimeric gene according to the invention may advantageously be composed of at least one promoter gene or promoter fragment that is naturally expressed in plants (tubulin, introns, actin, histone). The transcription termination signal region not translated at the 3 'end of the chimeric gene can be of any origin, for example of bacterial origin, such as that of the nopaline synthase gene, or of plant origin, such as that of the gene of H4A748 of histone from Arabidopsis t a-liana according to the European Patent Application (European Application 633 317). The chimeric gene according to the invention may comprise, in addition to the essential portions above, at least one non-translational intermediate region (linker), which may be located between the different transcribed regions described above. This intermediate region can be of any origin, for example of bacterial, viral or plant origin. Isolation of a cDNA that codes for a corn EPSPS The different steps that lead to the obtaining of corn EPSPS cDNA, which served as a substrate for the introduction of the two mutations, are described below.

All the operations described below are given via example, and correspond to a selection made from among the different methods available to reach the same result. This selection has no effect on the quality of the result, and consequently any suitable method can be used by a person skilled in the art to arrive at the same result. Most of these methods of DNA fragment construction are described in "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel FM et al., Published by Greene Publishing Associates and Wiley-Interscience (1989) (hereinafter referred to as reference to the protocols described in this work will be designated "CPMB ref."). The operations related to DNA that were performed according to the protocols described in this work are especially the following: DNA fragment binding, treatment with Klenow DNA polymerase and T4 DNA polymerase, preparation of plasmid and bacteriophage? DNA, either as a minipreparation or as a maxipreparation, and analysis of DNA and RNA according to Southern and Northern techniques, respectively. Other methods described in this work were followed, and only significant modifications or additions to these protocols have been described below.

Example 1: 1. Obtaining an EPSPS fragment from Arabidopsis thaliana a) Two 20-mer oligonucleotides of the respective sequences: 5 '-GCTCTGCTCATGTCTGCTCC-3' 5 '-GCCCGCCCTTGACAAAGAAA-3' were synthesized from the sequence of a EPSPS gene from Arabidopsis thaliana (Klee, HJ et al (1987) Mol. Gen. Genet., 210, 437-442). These two oligonucleotides are in positions 1523 to 1543 and 1737 to 1717, respectively, of the published sequence, and in opposite orientations. b) Total DNA from Arabidopsis thaliana (var. columbia) was obtained from Clontech (catalog reference: 6970-1). c) 50 nanograms (ng) of DNA were mixed with 300 ng of each of the oligonucleotides, and subjected to 35 cycles of amplification with a Perkin-Elmer 9600 apparatus, under the conditions of standard medium for amplification that are recommended by the supplier. The resulting 204 bp fragment constitutes the EPSPS fragment of Arabidopsis thaliana. 2. Construction of a cDNA library of a BMS maize cell line a) 5 g of filtered cells were ground in liquid nitrogen, and the total nucleic acids were extracted according to the method described by Shure et al. With the following modifications: the pH of the lysis buffer was adjusted to a pH of 9.0; - after precipitation with isopropanol, the pellet was dissolved in water and, after dissolution, adjusted to 2.5 M in LiCl. After incubation for 12 hr to ° C, the pellet of the centrifugation for 15 minutes at 30,000 g at 4 ° C was again solubilized. The precipitation step with LiCl was then repeated. The pellet re-solubilized constitutes the RNA fraction of the total nucleic acids. b) The poly (A) * RNA fraction of the RNA fraction was obtained by chromatography on an oligo (dT) -cellulose column, as described in "Current Protocols in Molecular Biology". c) Synthesis of double-stranded cDNA, which has one end of synthetic EcoRI: this is carried out in accordance to the protocol of the supplier of the different reagents necessary for this synthesis in the form of a case: the "copy case" of the company In Vitrogen. Two oligonucleotides of a single strand, and partially complementary to the respective sequences: 5'-AATTCCCGGG-3 '5'-CCCGGG-3' (the latter is phosphorylated) were ligated with the double-stranded sticky-end cDNAs. This ligation of the adapters results in the creation of S sites attached to the double-stranded cDNAs, and EcoRI sites in a cohesive form at each end of the double-stranded cDNAs. d) Creation of the library: The cDNAs that possessed the artificial cohesive EcoRI sites at their ends were ligated with bacteriophage cDNA, which had been cut with EcoRI and dephosphorylated according to the protocol of the supplier New England Biolabs. An aliquot of the ligation reaction was encapsidated in vitro with encapsidation extracts, ie Gigapack Gold, according to the supplier's instructions; this library was evaluated using the E. coli C600hfl bacterium. The library obtained by this means was amplified and stored according to the instructions of the same provider, and constitutes the cDNA library of BMS corn cell suspension. 3. Selection of the BMS maize cell suspension cDNA library with the EPSPS probe of Arabidopsis thaliana The protocol followed is that of "Current Protocols in Molecular Biology" Volumes 1 and 2, Ausubel, FM et al., Published by Greene Publishing Associates and Wiley-Interscience (1989) (CPMB). Briefly, approximately 10 € recombinant phages were seeded on plates, on LB discs, at an average density of 100 phages / cm2. The lithic plates were replicated in duplicate on Amersham Hybond N membranes. The DNA was fixed to the filters by UV treatment of 1600 kJ (Stratagene Stratalinker). The filters were pre-hybridized in 6xSSC / 0.1% SDS / 0.25 skim milk for 2 hours at 65 ° C. The EPSPS probe from Arabidopsis thaliana was labeled with [32 P] dCTP by randomizer addition, according to the supplier's instructions (Pharmacia Ready to Go case). The specific activity obtained is of the order of 10? cpm per μg of fragment. After denaturation for 5 minutes at 100 ° C, the probe was added to the prehybridization medium, and Hybridization was continued for 14 hours at 55 ° C. Filters were subjected to fluorography for 48 hours at -80 ° C with a Kodak XAR5 film and RPN increment screens from Amersham Hyperscreen. The alignment of the positive spots on the filter with the discs from which they originated allows areas corresponding to the phages that exhibit a positive hybridization response with the EPSPS probe of Arabidopsis thaliana to recover from the disk. This step of plating, transfer, hybridization and recovery is repeated until all the spots on the disk of the phage purified successively were 100% positive in the hybridization. An independent plate of phage lysis was recovered in medium? diluent (Tris-Cl pH 7.5, 10 mM MgSO4, 0.1 M NaCl, 0.1% gelatin); these phages in solution constitute the EPSP positive clones of the BMS corn cell suspension. 4. Preparation and analysis of the DNA of the EPSP clones of the BMS corn cell suspension. Approximately 5 x 108 phages were added to 20 ml of C600hfl bacteria, at a D060on value of 2 / ml, and incubated for 15 minutes. minutes at 37 ° C. This suspension was then diluted in 200 ml of bacterial growth medium, in a 1 liter Erlenmeyer flask, and shaken on a shaker rotating at 250 rpm. The lysis is noted when the medium is clarified, corresponding to the lysis of the turbid bacteria, and takes place after approximately 4 hours of agitation. This supernatant was then treated as described in "Current Protocols in Molecular Biology". The DNA obtained corresponds to the EPSP clones of the BMS corn cell suspension. One to two μg of this DNA were cut with EcoRI, and separated on agarose gel with 0.8% LGTA / TBE (ref. CPMB). A final check is to verify that the purified DNA indeed exhibits a hybridization signal with the EPSPS probe of Arabidopsis thaliana. After electrophoresis, the DNA fragments were transferred onto Amersham Hybond N membranes according to the Southern protocol described in "Current Protocols in Molecular Biology". The filter was hybridized with the EPSPS probe of Arabidopsis thaliana according to the conditions described in section 3 above. The clone exhibiting a hybridization signal with the EPSPS probe of Arabidopsis thaliana, and containing the largest EcoRI fragment has an estimated gel size of approximately 1.7 kbp.

. Obtaining clone pRPA-ML-711 Ten μg of phage clone containing the 1.7 kbp insert was digested with EcoRI, and separated on agarose gel with 0.8% LGTA / TBE (ref.CPMB). The gel fragment containing the 1.7 kbp insert was cut out of the gel by BET staining, and the fragment was treated with β-agarase according to the supplier's protocol, New England Biolabs. The purified DNA of the 1.7 kbp fragment was ligated at 12 ° C for 14 hours with the pUC 19 plasmid DNA (New England Biolabs) cut with EcoRI according to the ligation protocol described in "Current Protocols in Molecular Biology". Two μl of the above ligation mixture was used for the transformation of an aliquot of electrocompetent E. coli DH10B; the transformation was achieved by electroporation, using the following conditions: the mixture of electrocompetent bacteria and the binding medium were introduced in an electroporation cell with a thickness of 0.2 cm (Biorad) previously cooled to 0 C. The physical conditions of electroporation , using an electroporator made by Biorad are 2500 volts, 25 μF and 200 O. Under these conditions, the average discharge time of the capacitor is of the order of 4.2 milliseconds. The bacteria were then suspended in 1 ml of SOC medium (ref: CPMB), and stirred for 1 hour at 200 rpm on a rotary shaker in 15 ml Corning tubes. After plating on LB medium / agar supplemented with 100 μg / ml carbenicillin, minipreparations of the bacterial clones that had grown after overnight at 37 ° C were produced, according to the protocol described in "Current Protocols in Molecular Biology" " After digestion of the DNA with EcoRI and separation by electrophoresis on an agarose gel with 0.8% LGTA / TBE (ref: CPMB), the clones possessing a 1.7 kbp insert were retained. A final check is to verify that the purified DNA actually exhibits a hybridization signal with the Arabidopsis thaliana probe. After electrophoresis, the DNA fragments were transferred onto Amersham Hybond N membranes, according to the Southern protocol described in "Current Protocols in Molecular Biology". The filter was hybridized with the Arabidopsis thaliana probe according to the conditions described in section 3 above. The clone of the plasmid which possessed a 1.7 kbp insert, and hybridized with the EPSPS probe of Arabidopsis thaliana was prepared on a larger scale, and the DNA that resulted from the lysis of the bacteria was purified on a CsCl gradient as is described in "Current Protocols in Molecular Biology". The purified DNA was determined partially sequencing with a Pharmacia kit, according to the supplier's instructions, and using the M13 direct and reverse universal initiators ordered from the same supplier as initiators. The partial sequence produced covers approximately 0.5 kbp. The amino acid sequence derived in the region of the mature protein (approximately 50 amino acid residues) exhibits 100% identity with the corresponding amino acid sequence of the EPSPS of the mature corn described in US Pat. No. 4,971,908. This clone, which corresponds to an EcoRI fragment of 1.7 kbp of the EPSPS DNA of the BMS corn cell suspension, was designated pRPA-ML-711. The complete sequence of this clone was determined in both strands, using the protocol of the Pharmacia kit, and synthesizing complementary oligonucleotides, and those of the opposite orientation every 250 bp approximately. The complete sequence obtained from this 1713 bp clone is presented in SEQ ID No. 1. 6. Obtaining the clone pRPA-ML-715 The analysis of the sequence of the clone pRPA-ML-711, and especially the comparison of the sequence of amino acid derived with that of corn, shows a sequence extension of 92 bp upstream of the GCG codon encoding the NH2-terminal alanine of corn EPSPS (US Patent No. 4 USP971.908). Similarly, an extension of 288 bp downstream of the AAT codon encoding the COOH terminal asparagine of the mature portion of the corn EPSPS is observed (US Patent No. 4,971,908). These two portions could correspond, in the case of the NH2-terminal extension, to a portion of the sequence of a transit peptide for the location of the plastid, and, in the case of the terminal COOH extension, to the 3 'region not Translated from the cdn. To obtain a cDNA coding for the mature portion of the maize EPSPS cDNA, as described in USP No. 4,971,908, the following operations were carried out: a) Elimination of the 3 'untranslated region: construction of pRPA- ML-712: The clone pRPA-ML-711 was cut with the restriction enzyme Asel, and the ends that resulted from this hydrolysis became sticky by treatment with the Klenow fragment of DNA polymerase I according to the protocol described in CPMB. A hydrolysis was then carried out with the restriction enzyme SacII. The DNA resulting from these operations was separated by electrophoresis on agarose gel with 1% LGTA / TBE (ref: CPMB).

The gel fragment containing the 0.4 kbp "Asel-sticky ends / SacII" insert was cut from the gel, and purified according to the protocol described in section 5 above. The DNA of the clone pRPA-ML-711 was cut with the restriction enzyme HindIII at the HindIII site located in the polylinker of the cloning vector pUC19, and the resulting ends of this hydrolysis became sticky by treatment with the Klenow fragment of the DNA polymerase I. Lego hydrolysis was performed with the restriction enzyme SacII. The DNA resulting from these manipulations was separated by electrophoresis on an agarose gel with 0.7% LGTA / TBE (ref: CPMB). The gel fragment containing the insert of approximately 3.7 kbp of HindIII-sticky ends / SacII was cut from the gel, and purified according to the protocol described in section 5 above. The two inserts were ligated, and 2 μl of the ligand mixture was used to transform E. coli DH10B, as described above in section 5. The plasmid DNA content of different clones was analyzed according to the procedure described for pRPA -ML-711. One of the selected plasmid clones contained an EcoRI-HindIII insert of approximately 1.45 kbp. The sequence of the terminal ends of this clone revealed that the 5 'end of the insert corresponds exactly to the corresponding end of pRPA-ML-711, and that the 3' end has the following sequence: "5 '-" AATTAAGCTCTAGAGTCGACCTGCAGGCATGCAAGCTT-3' The underlined sequence corresponds to the codon of the amino acid asparagine COOH terminal, the next codon corresponds to the translational stop codon.The downstream nucleotides correspond to sequence elements of the pUC19 polylinker.This clone, which comprises the sequence pRPA-ML-711 to the translation termination site of the EPSPS of mature corn, and followed by sequences of the polylinker pUC 19 to the HindIII site was designated pRPA-ML-712. b) Modification of the 5 'end of pRPA-ML-712: construction of pRPA-ML-715: The clone pRPA-ML-712 was cut with the restriction enzymes PstI and HindIII, the DNA resulting from these manipulations was separated by electrophoresis on agarose gel with 0.8% LGTA / TBE (ref. CPMB). Aggregate gel containing the 1.3 kbp PstI-EcoRI insert was cut from the gel and purified according to the protocol described in section 5 above. This insert was ligated in the presence of an equimolecular amount of each of the two partially complementary sequence oligonucleotides: Oligo 1: 5 '-GAGCCGAGCTCCATGGCCCGGCGCCGAGGAGATCGTGCTGCA-3' Oligo 2: 5'-GCACGATCTCCTCGGCGCCGGCCATGGAGCTCGGCTC-3 'as well as in the presence of plasmid pUC19 DNA digested with the restriction enzymes BamHl and HindIII. Two μl of the binding mixture was used to transform E. coli DH10B as described above in section 5. After analysis of the DNA content of the plasmid of different clones according to the procedure described above in section 5, one of the clones, which had an insert of approximately 1.3 kbp was retained for subsequent analysis. The sequence of the 5'-ter end of the selected clones revealed that the DNA sequence in this region is as follows: sequence of the pUC19 polylinker from the EcoRI to BamHI sites, followed by the sequence of the oligonucleotide used in the cloning, followed by the remainder of the sequence present in pRPA-ML-712. This clone was designated pRPA-ML-713. This clone possesses an ATG codon of methionine included in a Ncol site upstream of the N-terminal alanine codon of the mature EPSPS synthase. Additionally, the alanine and glycine codons of the N-terminal end have sid preserved, but modified on the variable third base: the initial GCGGGT gives the modified GCCGGC. The clone pRPA-ML-713 was cut with the restriction enzyme HindIII, and the ends of this hydrolysis became sticky by treatment with the Klenow fragment of DNA polymerase I. A hydrolysis was then performed with the restriction enzyme Sacl. The DNA that resulted from these manipulations was separated by electrophoresis on an agarose gel with 0.8% LGTA / TBE (ref: CPMB). The gel fragment containing the "HindIII-sticky ends / Saci" insert of 1.3 kbp was cut from the gel and purified according to the protocol described in section 5 above. This insert was ligated in the presence of plasmid pUC19 DNA, digested with the restriction enzyme Xbal, and the ends of this hydrolysis became sticky by treatment with the Klenow fragment of DNA polymerase I. A hydrolysis was then performed with the restriction enzyme Sacl. Two μl of the binding mixture was used to transform E. coli DH10B as described above in section 5. After analysis of the DNA content of the plasmid of different clones according to the procedure described above in section 5, one of the clones, which had an insert of approximately 1.3 kbp was retained for analysis subsequent The sequence of the terminal ends of the selected clones revealed that the DNA sequence is as follows: polylinker sequence of pUC19 from the EcoRI to Sacl sites, followed by the sequence of the oligonucleotides used in the cloning, of which the GATCC 4 bp of the oligonucleotide 1 described above has been deleted, followed by the remainder of the sequence present in pRPA-ML-712 to the HindIII site, and the polylinker sequence pUC19 from Xbal to HindIII. This clone was designated pRPA-ML-715. 7. Obtaining a cDNA that codes for an EPSPS of mutated corn All the stages of the mutagenesis were carried out with the mutagenesis kit of Pharmacia U.S.E. according to the supplier's instructions. The principle of this mutagenesis system is as follows: the plasmid DNA is denatured by heat, and it is re-associated in the presence of a molar excess of, on the one hand, the mutagenesis oligonucleotide, and on the other hand an oligonucleotide that allows that a single restriction enzyme site present in the polylinker be removed. After the annealing step, the synthesis of the complementary strand was carried out by the action of T4 DNA polymerase, in the presence of T4 DNA ligase and 32 gene protein in a suitable buffer solution that is supplied. The product of the synthesis was incubated in the presence of the restriction enzyme for which it is assumed that the site has disappeared by mutagenesis. The strain of E. coli which possesses, in particular, the mutS mutation is used as a host for the transformation of this DNA. After culture in liquid medium, the total plasmid DNA was prepared and incubated in the presence of the restriction enzyme used before. After these treatments, the E. coli strain DH10B was used as the host for the transformation. Plasmid DNA was prepared from the isolated clones, and the presence of the introduced mutation was verified by sequence determination. A) - modification of sequence sites without in principle affecting the EPSPS resistance character of maize to products that are competitive inhibitors of EPSPS synthase activity: elimination of an internal Ncol site from pRPA-ML-715. The sequence of pRPA-ML-715 was numbered arbitrarily, placing the first base on the GCC codon of N-terminal alanine at position 1. This sequence possesses a Ncol site at position 1217. The oligonucleotide with site modification possesses the sequence: 5'-CCACAGGATGGCGATGGCCTTCTCC-3 '.

After determining the sequence according to the references given above, the sequence read after the mutagenesis corresponds to that of the oligonucleotide used. The Ncol site has actually been removed, and translation into amino acids in this region preserves the initial sequence present in pRPA-ML-715. This clone was designated pRPA-ML-716. The 1340 bp sequence of this clone is presented in SEQ ID No. 2, and SEQ ID No. 3. B) sequence modifications that allow the EPSPS resistance character of maize to be increased to products that are competitive inhibitors of the EPSPS synthase activity. The following oligonucleotides were used: a) Thr 102 mutation? He. 5 '-GAATGCTGGAATCGCAATGCGGCCATTGACAGC-3' b) Pro 106 mutation? Ser. 5 '-GAATGCTGGAACTGCAATGCGGTCCTTGACAGC-3' c) Gly 101 mutations? Ala and Thr 102? He. 5 '-CTTGGGGAATGCTGCCATCGCAATGCGGCCATTG-3' d) Thr 102 mutations? He and Pro 106? Ser. 5 '-GGGGAATGCTGGAATCGCAATGCGGTCCTTGACAGC-3' After determination of the sequence, the sequence read after mutagenesis on the three mutated fragments is identical to the parent pRPA-ML-716 sequence, with the exception of the mutagenized region corresponding to that of the mutagenesis oligonucleotides used. . These clones were designated: pRPA-ML-717 for the Thr 102 mutation? He, pRPA-ML-718 for the Pro 106 mutation - > Ser, pRPA-ML-719 for Gly 101 mutations? Ala and Thr 102? He and pRPA-ML-720 for Thr 102 mutations? He and Pro 106 - • Ser. The 1340 bp sequence of pRPA-ML-729 is presented in SEQ ID No. 4 t SEQ ID No. 5. The 1395 bp NcoI-HindII insert is the basis of all constructions used for the transformation of silvers for the introduction of resistance to herbicides that are competitive inhibitors of the EPSPS, and especially the resistance to glyphosate. This insert will be designated in the remainder of the description "the maize EPSPS double mutant". Example 2: Tolerance to glyphosate of different in vitro mutants 2.a: Extraction of EPSPS synthase The different EPSPS synthase genes were introduced in the form of a NcoI-HindIII cassette in the plasmid pTrc99a vector (Pharmacia, ref: 27-5007-01) cut with Ncol and HindIII. Recombinant E. coli DH10B bacteria, which overexpressed the different EPSPS synthases were sonicated in 40 ml of buffer solution per 10 ml of cells as pellets, and washed with this same buffer (200 mM Tris-HCl pH 7.8, 50 mM in mercaptoethanol, 5 mM in EDTA and 1 mM in PMSF), to which 1 g of polyvinyl pyrrolidone was added. The suspension was stirred for 15 minutes at 4 ° C, and then subjected to centrifugation for 20 minutes at 27,000 g and 4 ° C. Ammonium sulfate was added to the supernatant to bring the solution to 40% saturation with respect to ammonium sulfate. . The mixture was subjected to centrifugation for 20 minutes at 27,000 g and 4 ° C. Ammonium sulfate was added to the new supernatant, to bring the solution to 70% saturation with respect to ammonium sulfate. The mixture was subjected to centrifugation for 30 minutes at 27,000 g and 4 ° C. The EPSPS synthase present in this protein pellet was suspended in 1 ml of buffer solution (20 mM in Tris-HCl pH 7.8 and 50 mM in mercaptoethanol). This solution was dialyzed overnight against two liters of this same buffer at 4o C. 2. b: Enzyme activity The activity of each enzyme, as well as its resistance to glyphosate, was measured in vitro for 10 minutes at 37 ° C in the following reaction mixture: 100 mM in maleic acid pH 5.6, 1 mM in phosphoenolpyruvate, 3 mM in shikimate 3-phosphate (prepared according to Knowles, PF and Sprinson, DB 1970. Methods in Enzymol 17A, 351-352 from Aerobacter aerogenes strain ATCC 25597) and 10 mM in potassium fluoride. The enzyme extract was added at the last moment, after the addition of glyphosate, the final concentration of which varied from 0 to 20 mM. The activity was measured by assaying the phosphate released according to the technique of Tausky, H.A. and Shorr, E. 1953. J. Biol. Chem., 202, 675-685. Under these conditions, the wild type enzyme (WT) is already 85% inhibited at a glyphosate concentration of 0.12 mM. At this concentration, the mutant enzyme known as Serl06 is only 50% inhibited, and the other three mutants, Hel02, Hel02 / Serl06 and Alal01 / Hel02, show little or no inhibition. The concentration of glyphosate has to be multiplied by ten, that is 1.2 mM, to produce a 50% inhibition of the mutant enzyme Hel02, mutants Hel02 / Serl06, Ala / He and Ala are not yet inhibited. It should be noted that the activity of the Ala / He and Ala mutants is not inhibited up to the glyphosate concentrations of 10 mM, and that that of the mutant Ilel02 / Serl06 is not reduced even if the concentration of glyphosate is multiplied by 2, that is, by say 20 mM. Example 3: Resistance of transformed tobacco plants 1-1 Transformation Vector pRPA-RD-173 was introduced into Agrobacterium tumefaciens strain EHA101 (Hood et al., 1987) carrying the cosmid pTVK291 (Komari et al., 1986). The transformation technique was based on the procedure of Horsch et al. (1985). 1-2 Regeneration the regeneration of tobacco PBD6 (source SEITA France) from leaf explants was carried out on a basal medium of Murashige and Skoog (MS), comprising 30 g / liter of sucrose, as well as 200 μg / ml of kanamycin. The leaf explants were extracted from plants grown in the greenhouse or in vitro, and were transformed according to the leaf disc technique (Science, 1985, Vol. 227, pages 1129-1231) in three successive stages: Prinera comprises the induction of buds on a medium supplemented with 30 g / liter of sucrose, which contained 0.05 mg / liter of naphthylacetic acid (NAA) and 2 mg / liter of benzylaminopurine (BAP) for 15 days. The buds formed during this stage were then grown for 10 days by growing them on an MS medium supplemented with 30 g / l sucrose., but that did not contain any hormones. The buds that were developed were then extracted, and were grown on an MS root medium that had half the content of salts, vitamins and sugar, and did not contain any hormones. After about 15 days, the yolks with rootlets were transferred to soil. 1-3 Resistance to glyphosate Twenty transformed plants were regenerated and transferred to the greenhouse for the construction of pRPA-RD-173. These plants were treated in the greenhouse at the 5 leaf stage with an aqueous suspension of RoundUp corresponding to 0.8 kg of glyphosate active substance per hectare. The results correspond to the observation of phytotoxicity indexes registered 3 weeks after treatment. Under these conditions, it was found that the plants transformed with the pRPA-RD-173 construction they exhibit a very good tolerance, while the non-transformed control plants are completely destroyed. These results clearly show the improvement brought about by the use of a chimeric gene according to the invention for the same gene that codes for tolerance to glyphosate. Example 4: Transformation and selection of corn cells BMS corn cells (Black Mexican Sweet), in an exponential growth phase were bombarded with the construction of pRPA-RD-130 according to the principle and the protocol described by Klein et al. 1987 (Klein, T.M., Wolf, E.D., Wu, R. and Sandford, J.C. (1987): High velocity microprojectiles for delivering nucleic acids into living cells, NATURE Vol. 327, pages 70-73). Two days after the bombardment, the cells were transferred to the same medium, which contained 2 mM N- (phosphonomethyl) glycine. After 8 weeks of selection on this medium, the calluses that developed were selected, then amplified and analyzed by PCR, and clearly revealed the presence of the chimeric OTP-EPSPS gene. Cells that were not bombarded and cultured on the same medium containing N-. { phosphonomethyl) glycine 2 mM were blocked by the herbicide, and did not develop. The plants transformed according to the invention can be used as parents to obtain lines and hybrids having the phenotypic character corresponding to the expression of the gene. chimeric introduced. Description of plasmid constructions pRPA-RD-124: Addition of a polyadenylation signal "nos" to pRPA-ML-720 with creation of a cloning cassette containing the double mutant EPSPS maize gene. { Thr 102 ? He and Pro 106? Be) . PRPA-ML-720 was digested with HindIII, and treated with the Klenow fragment of E. coli DNA polymerase I to produce a sticky end. A second digestion was performed with Ncol, and the EPSPS fragment was purified. The EPSPS gene was then ligated with purified pRPA-RD-12 (a cloning cassette containing the polyadenylation signal of nopaline synthase) to give pRPA-RD-124. To obtain the useful purified pRPA-RD-12 vector, it was necessary that the latter be previously digested with Sali, treated with Klenow DNA polymerase, and then digested a second time with Ncol. PRPA-RD-125: Addition of an optimized transit peptide (OTP) to pRPA-RD-124 with creation of a cloning cassette containing the EPSPS gene targeted on the plasmids PRPA-RD-7 (European Patent Application EP 652 286) was digested with Sphl, treated with T4 DNA polymerase, and then digested with Spel, and the OTP fragment was purified. This fragment of OTP was cloned into pRPA-RD-124, which had previously been digested with Ncol, treated with Klenow DNA polymerase to remove the 3 'portion that protruded, and then digested with Spel. The sequence was then determined to this clone, to ensure the correct translational fusion between the OTP and the EPSPS gene. Then pRPA-RD-125 was obtained. pRPA-RD-130: Addition of the H3C4 corn histone promoter and the adhl intron 1 sequences of pRPA-RD-123 (Patent Application EP 507 698) to pRPA-RD-125 with creation of a cassette for the expression in plants, for the expression of the double mutant EPSPS gene in monocotyledon tissues. pRPA-RD-123 (a cassette containing the corn histone H3C4 promoter fused with the inhl 1 of adhl) was digested with Ncol and Sacl. The DNA fragment containing the promoter derived from pRPA-RD-123 was then purified, and ligated with pRPA-RD-125, which had previously been digested with Ncol and Sacl. pRPA-RD-159: Addition of the double histone promoter of Arabidopsis H4A748 (Patent Application EP 507 698) to pRPA-RD-125 with creation of a cassette for expression in plants, for the expression of the "OTP-gene of double mutant EPSPS gene" in the tissues of dicotyledonia. pRPA-RD-132 (a cassette containing the double promoter H4A748 (Patent Application EP 507 98)) was digested with Ncol and Sacl. The purified promoter fragment was then cloned into pRPA-RD-125, which had been digested with Ecol and Sacl. pRPA-RD-173: Addition of the gene "promoter H4A748-OTP-double mutant EPSPS gene" from pRPA-RD-159 to plasmid pRPA-RD-150A (European Patent Application 508 909) with creation of a. transformation vector of Agrobacterium tumefaciens. pRPA-RD-159 was digested with Notl, and treated with Klenow polymerase. This fragment was then cloned into pRPA-BL-150A with S al.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTS: Lebrun, Michel Freyssinet, Georges Sailland, Alain (ii) TITLE OF THE INVENTION: mutated 5-enol pyruvyshikimate-3-phosphate synthase, gene that codes for this protein, and Transformed plants that contain the gene. (iii) NUMBER OF SEQUENCES: 5 (iv) DOMICILE FOR CORRESPONDENCE: (A) RECIPIENT: Francois Chretien (B) ADDRESS: 1420 rue de Pierre baizet (C) CITY: Lyon Cedex 09 (E) COUNTRY: France (F) CODE POSTAL: 69263 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMMING ELEMENTS: Patentln Reléase # 1.0, Version # 1.25 (vi) DATA OF THE PRESENT APPLICATION: (A) NUMBER OF THE APPLICATION: (B) SUBMISSION DATE: (C) CLASSIFICATION: (VIII) INFORMATION OF THE POWDER / AGENT (a) NAME: Chretien, Francoise (IX) TELECOMMUNICATION INFORMATION (A) TELEPHONE: (33) 72-29-26-46 ( B) TELEFAX: (33) 72-29-28-43 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1713 base pairs (B) TYPE: nucleic acid ( C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Zea mays (B) strain: Black Mexican Sweet (C) TYPE OF TISSUE: Callo (vii) IMMEDIATE SOURCE: (A) LIBRARY: lambda gtlO (B) CLON: pRPA-ML-711 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 AMCMirt jbacMßM? cMcuaoie aaw-ruco jutrranooe aocucBCSTß «0 120 ISO 210 300 M0 420 C? ßTtACTOC IlCTOCTW- »AMOCM TG JXJimem TaeMRM3-? JafiM? WßQO 100 $ 40 TCCTTCCCAC TC? CTKpt? Ltt nuftc TC? A CfiAAT CGSAOGGCTA ccrsctsocA ßoo AGCTG ?? GCT CTCTCO UJ ATCAGCATGTC A6r * crrc «toerrrcctc ATpfictcctc« M C? TTßOCTCT TGQO? TCTG WBATP? AAA TCATTCATAA A.TTAA: TrCT ATTCSTAOB JÍ0 TOBAAATCA ATTSMATTr; ATOCOCTT T7QCTOTCAA ACCMMBCKT T TGATACCT 7ß0 CCßAOnATT TACATTAAG CSAßßTCMA A? TACMCTC CCCGJ AKAT GCCTATCTTC • 40 AACCTGATCC CTC CaeC *. ACCTATTICT TOO TGGTOC COA1 AC7 0BMBCSM7IC 900 M0 1020 1000 1140 1200 1200 1320 1300) 440 isoo L.I I ...? .. c.ii Acscc ATTA? CTGTG OM? CTGTAA CCTTAU? UI? CTAGCMC 11 * 0 1 ! H.IATTTC CSTATTMAY 7STACT »CT CTM0CCM? 1.A. X? JUMSTGCTTC IC20 GTTGGMT ?? TA? C? ATAA? ??? TTAC-TTT TOIGT6AAAA AAAAAA ?? AA AAAAAAAAAA. «• 0 1113 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1340 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Zea ays (B) strain: Black Mexican Sweet (C) TYPE OF TISSUE: Callus (vii) IMMEDIATE SOURCE: (B) CLON: pRPA-ML -716 (ix) ASPECTS: (A) NAME / KEY: CDS (B) POSITION: 4 ... 1337 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 ec OM OM Ate oro ere XTC AM GM ATC 4T Al * ßly Wing Olw ßlw Xla Val Lmt ßl * * t aunt Lr * ßlu Z a 1 s 10 TOC OQC ACC ßTC AM CW Oaj ßOß TCCAM TOB Crr TOC AAC CS6 ATC ts tme ßlr Thr V l Lfw Lau ff »ßlr aVor Lr * | * r Lau Oar Aan Ara lia i »2ß xs ao ere CTA CTC ßcc occ ero tec CAO ose ACÁ ACÁ OT OT 8 AAC ese J Lau Lau Law Ala Ala Lau »ar Olw € Xf Tkr Thr Val Val Aaa Aan Lau» 40 4 > ere AAC AOT CM CAT ore sc TAC ATS ere oaß ßcc TTB MS Acr CTT iti Lau A * n Oar 61M Aaa > Val N a Tyr Ha * Lau ßlr Ala Lau Ara Th * Lau 10 »» * ß c.tt CTC? Rt CTC CAÁ cas CAC AAA OCT GCC AAA AC CCT CTA srt CTT JI Cir »$ *» * •• C At * ?? t > t, rt Ai »* e» * rg AI * V »Í V.« I JI ib »0 7 * GT see t¡m w *?« ttc? Acrrr cA * eAT GC AAA CAc cAA trc cC «> ctr Crc ciy ßiy cr * "» • • »» »Vßl Ci" * - "" ** • LV «Gl" Ciu v * 1 ln 40 SS 40 A OR TTC ACÁ CCA CCT 335 CTC TTC Tr. CCC AAT CCT CSA ACT CCA CSS CC Thr Ala Ala n Ala Cly Tnr Al * Mat Aura rro Law Lau fhß Lau Cly Aa IOS UO 15 100 TS CTT CAT OSA CTA OCA 303 CTT ACT CCT CCT CCT OCA AAT OCA ACT TM G v »? Tnr Ala Ala ßly ßly Aan Ala Thr Tyr Val Law Aap Cly val rrß US 120 431 ACÁ ATC ACC CM ACÁ CCC ATT CSC CM TTC 6TT Arg Nat Ara Clw Ara fro? L * 61and no Uu Val 120 135 A CCT CTT 47t CTT GGT CCA CAT CTT CAT TCT TTC CTT CSC ACT CAC TCC CC already rhß Law ßly Thr Aap cya rro reo V Lag ßly Wing Aßp Val Aßp C at 14S ISO US CCC AM ßTC AM CTO TCT 527 CCT GTC AAT 06A? TC CCA CßC CTA CCT CST Ara Val Aan ßly Xla Cly ßly Law rrß ßly ßly Ly »Val Lyß Law Ser ICO 1SS 170 CCC TTC CTC ATC CCT CCT CCT 575 OOC CC ATC ACC MT CM TAC TTC ACT Sar Sar ßlrt Tyr Law aar Ala Lau Lau Mat Ala? La rro ßly Sar 11 * 190 PS 100 íes CAT GTC CM ATT CAÁ ATC ATT CAT AAA TT? ATC TCC «23 TTC ß r CTT CCS Uu Wing Law ßly Am »Val ßlw Zl * ßlu Zla Zlß Aap Lyß Lau lia Sar 1 * S 200 205 C CAÁ ATS ACÁ TTS AGA TTC ATC CM CCT TTT CCT ere« 71 ATT CCS TA lia rro Va T Thr Law Arq Uu Mat Clu Ara rrtß ßly l yr val Clu Nat 210 21S 220 ASA TTC TAC ATT AM ßßA CCT Til AAA CCA CAC C? T CT 6AT MC TOß CM Lft Ala Clu Mía Sar Aßp Ser Tr »Aa» Ara Pha Tyr Zla Lyß ßly ßly 22S 220 235 r AAA AAT OCC TAT CTT CAÁ 6ßT CAT CCC TCA 7 «1 CAA AAA TM AM TCC er Sin Lf »Tyr Lya Sar rra Lya Aan Wing Val ßlw Cly Aßp Wing E 140 24S 250 ACC CCA ACC TAT TC TTC OCT CCT OCT OCA ATT ACT OCA CCC ACT GTC • 15 Sar Ala Sar Tyr m * Law Ala ßly Ala Ala Zla Thr Cly ßly Thr at 255 200 2 «S 270 ACT CTC CAÁ CCT TCT CCC ACC ACC MT TTß CM CCT CAT GTC AM TTT • < 3 Thr val ßlw Cly Cya ßly Thr Thr Sar UN ßLn ßly Aa »Val Lyß rhß 200 20S OCT CAC CTA ere CM ATC ATO CCA ees AM ßTT ACÁ TOß ACC ßM ACT 111 Ala ßlw Val Law ßlw Mat Hat ßly Al * Lyß Val Thr Trp Thr ßlu Thr 200 MC CTA Mß AAA CAC 151 Car Val Ara .Lya Hiß 1007 CTC AM ATT CAT ere AM ATO AM AM ATO ccT CAT ere Law Lyß Ala Zla Aa »Val Aan Mat Aßn Lya Mat rra Aap Val Wing 220 220 SSS ACT CTT ecT CTß ßTT ere TTT ßcc CAT ßße CCS ACÁSOC ATC ASA 10SS Thr Lau Ala goes. Val Ala Law Sha Al * Aa »ßly rra Thr Ala Zla Ara SSO MS 240. 14 $ CM CTß CCT TCC TOß MA CTA AM ßM ACC OM ß ATO ßTT ßTB ßCB ATC 1103 Aaa Val Ala Sar Tra Ara Val Lyß ßlu Thr ßlu Ara Mal Val Ala tía 3SS MO MS OBB B ßM CTA MC AM CTß ßßA ßCA TCT ßTT ßM CAÁ ßßß CCO C? C 1131 Ara Thr ßlw Law Thr Lya Lau ßly Ala • me Val ßlu ßlu ßly "or Aap »» O m 300 t c -ro ATC AYC * cs ccc CCT CAC AAC AAC CTC AC: CCC ATC CM H M Tyr Cwlia CJ? R Thr * rro C Lyß Aan vn Thr AU U «Aa« • t CTT CCC «S TCT 1217 WT Lru Ala Al * Cy» 12tS *! • O.u v.i aro v.? Tnr Zl. Aro A. »rrß ßly Cy. Thr Ara Lyß Thr "4 * ° 425 430 fh * f ro.» Tyr fh. Aap Val Uu Se, Thr fr .. Val Lya Aan 435 «« or 7AA 1340 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 444 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Al * ßly Al * ßlu ßlu Zl * Val Law ßln rra? The Lyß ßlu Zlß Ser Cly 1 5 10 13 Thr Val Lya Uw rro ßly Sar Lya Ser Uu Ser Aan Ara Zlß Uu Uu 20 25 30 Uu Ala Ala Uw Ser ßlu ßly Thr Thr Val Val Aap Aan Lau Uu Aßn 35 40 45 Ser ßlu Aa »Val Ml * Tyr N * t Uu Cly Al * Uu Ara Thr Uu Cly Uu 50 55« OS * r val ßlu A * Aa »Ly * Al * Al * Lya Ara Al * Val Val Val ßly Cyß «I 0 15 SO ßly ßly Lyß me rß va l ßlu Aß» Wing Ly * ßlu ßlu Val ßln Lau me SS M 15 Uw ßly Aan Al * ßly Thr Ala Mat Aro fr Lau Lau Ala Ala Val Thr 100 OS 110 Ala Ala ßly ßly Aan Al Thr Tyr Val Uu Aap Cly Val rro Ara Mat 115 120 125 Ara ßlu Ara rro Zle ßly Aa »Uw Val Val ßly and Uu Lyß ßln Lau ßly 130 135 140 Ala Aap Val Aap Cyß fha Uw O and Thr Aßp Cyß rrß rrß Val Ara Val 145 150 155 1 «0 Aan ßly t a ßly ßly Uu rro ßly ßlr Lr * * • * Lya Law Sar ßly Mr 1 «S 170 17S Zlß Ser Sar ßln Tyr Uw Ser Ala Uw Uw Mat Ala Ala Lau Lau Ala 100 IOS ISO Law ßly Aßp Val ßlw Zlß ßlu Zlß Zlß Aßp Lyß Leu XI to Sar Zla rra 115 200 20S Tyr Val ßlw Mat Thr Uw Ara Uu Mat ßlw Ara rha ßly Val Lyß Ala 210 215 220 ßlw Hta be Aßp Ser Trp Aßp Ara rho Tyr Zlß Lyß ßly «and ßln Ly» 225 230 235 240 Tyr Lyß Ser rro Lye Aan Wing Tyr Val ßlu ßly Aßp Wing Be flower Wing 245 2S0 2SS Wtf Tyr fft * The »la Cly Ate Ala? Ie Thr Cly Cty Thr Val Thr Va | 1 * 0: «t 2nd AUClv C? Clr Thr Tnr Sor Uw Cln Cly Aap Val Lr- "he Al * Gw? 15 200 t» S < Vy Uu blu Met Mßt- Cly Ala L a al Thr Trp Thr Clu Thr Ser v «| l < * 0 «5 J? J Thr Val Thr ßt aro rro Ara Clu rro rn * Cly Ara Lys Hl * Uu Lys JO! 310 315 320 To * II. Aap val Aan Mat Aan Lya Hat rro Aap Val Ala Mßt Tur Uu 325 330 333 Wing goes: Val Al * Uw me Al * Aßp ßly rr Thr Ala? Ara Aap Val 140 34S 350 Al * Ser Trp Ara v * l Lyß ßlw Thr ßlu Ara Met Val? Zlß Arg Thx 3SS 300 MS ßlu Uu Tnr Ly * Uw Gly Ala Ser Val Clu Clu Clyro Aap Tyr Cya 370 37J 300: i * Zle Tnr fro rro Clu Lyß Uw Aan Val Thr Ala? The Aßp Thr Tyr J «l 3tO 39S 400 Aap Aap Mía Ara Mßt Ala Mßt Ala rhe Ser Uu Ala Ala Cyß Ala Clu 405 410 41S Val rro Va. Thr lia Ara Aßpro Cly cyß Thr Ara Lya Thr rho rro 420 425 430 Aap Tyr me Aà £ o Val Uu e? Thr a Val Lyß Aan (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1340 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Zea mays (B) strain: Black Mexican Sweet (vii) IMMEDIATE SOURCE: (B) CLON: pRPA-ML-720 (ix) ASPECTS: ( A) NAME / KEY: CDS (B) POSITION: 4 ... 1337 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 CCATß ßCC ßOC CCC CM CM ATC CTß CTß CM CCC ATC AM CM ATC 47 Ala ßlr Ala ßlw ßlu Zle Val Lau ßln rro lia? * Clu Zla * 5 10 TCC ccc ACC ere AM ero ees ßeß roe AM TCB CTT TCC AM that ATC ts Sor ßlr TTtr Val Ly * Lau tim ßlr Sor Lr »ßßr Lau Sor Aßn Ara Zla" 20 25 30 CTC CTA CTC ccc oce ero TOC ßM ßßß ACÁ ACÁ ere ott CAT AM CTC 143 Lau Ala Ala Law Ser ßlu ßlr Thr Thr Val Val Aßp Aßn Law 35 «0 45 CTO AM AßT CM CAT OTC CM TM ATß CTC eße ßOC TTO C ACT CTT ltl Ser ßlw Aap val Kla Tyr Met Law ßly Ala Lau Ara Tnx Lau 50 SS «0 ТT CTC TCT CTC CAA ßCS ßM AAA ßcr OCC AAA AflA OCT OTA ßTT OTT 23 S ßlr Lau Car Val ßlw Ala Aap Lyß Ala Ala Lr» Arg Ala Val Val Val ** 70 7S CeC TCT C «JT GCA AC TTÍ CCA CTT C * C CAT CCT AAA CAC CAÁ CTC CAC J» »1 C? C-? Cl y C l y \, t ** > • «re Va l C t-. ? ap Wing L »Clu Clu v« i Cln pO • * '• "0 CTC TTC TGC OCT"? T CCT CCA ATC CCA AT CCC rec rrc ACÁXA CCT 335? ra phita. Leu C ly AI? A l * C Xle Al * Met Aro Ser > w Th r A l «Al» qrs ion 1 1 CTT? CT CCT CCT CßT < WA AAT CCA ACT TAC TG CTT UAT OSA A CCA JOJ v «l Th Ala Ala Cy Cly Aan Ala Tnr Tyr al Lau Aßp tíly Val rro U * 120 1 = 5 ASA ATC ACC CAC MA CCC ATT CCC CAC TTC CTT CTC GCA TTS AM CM 431 Aro Mee Ara C w Arq rro Zl * Cly Aßp Leu Val Val Cly Lau Lyß Gln 130 133 140 CTT CCT CCA CAT CTT CAT TCT TTC CTT CeC ACT CAC TGC OCA CCT CTT 47 » Leu Cly Al * Aap Va l Aa »Cyß rhe Lau Cly Thr Aap Cy r rro Pro Val 1 130 155 CCT CTC AAT CGA ATC OCA ßßß CTA CCT CCT CßC AAC CTC AAC CTG TCT 527 Ara Val Aan Cly Zla Cly Cly Law Cly Cly Ly »Val Lyß Lau Ser 1« 0 ICS 170 eae TCC ATC AGC ACT CM TM TTC MT eee TTC CTC AG CCT CCT CCT 5? S Cly Ser Zla Sor sime ßln Tyr Lau »ßr Ala Lau Lau Met Ala Wing 17S 100 IOS 1S0 TTC CCT CTT cec CAT ere CAC ATT CAÁ ATC ATT GAT AAA TTA ATC TCC« 23 Lau Ala Lau Cly Aßp Val Clu Zla Clw Zla? La Aap Lya Lau Zla Sar ItS 200 205 ATT CCC TM CTC CAÁ ATß ACÁ TTC AßA TTC ATC CAC CCT TTT CCT CTC «T: i * rro Tyr val Clu Mßt Thr Law Ara Law Mat Clu A e Phe Cly Val 210 215 220 AAA CCA GAS CAT TCT CAT ACC TCC CAC AßA TTC TAC ATT AM CCA CCT US Lya Ala Clw Hla Sar Aap * ar Trp Aap Arq r e Tyr? Lß Lyo Gly Gly 225 230 235 CAÁ AAA TM AAC TCC CCC TAT CTT CAÁ CCT CAT CCC TCA 7 «7 Without Ly * Tyr Ly * Ser rro Ly * Aan Ala Tyr Val Clw ßly Aap Ala Sar 240 24S 250 ACC CCA MC TAT TTC TTS 6CT COT CCT SCA ATT ACT COA CCC ACT CTC 815 Be Al *% ee Tyr Pho uu Al * Cly Al * Al * Zl * Thr Cly Cly Thr Val rss 2S0 2CS 270 ACT 5TS CAOT COT TCT cec ACC ACC ACT rrc C CCT CAT CTC AM TTT sß3 Thr Val Clw Cly Cy * ßly Thr Thr S * r Law ßln Cly Aap Val Lyo rha 275 2S0 205 CCT CAC CTA CTß CAS ATC ATC CCA CCS AM CTT ACÁ TCC? CC CAC ACT Sil Al * ßlu Val Law ßlw Met Met ßly Wing Lya Val Thr Trp Thr Clu Thr 200 2S5 300 MC CTA ACT ßTT ACT CßC OCA CCS CBß CM OCA TTT COß AOC AAA CAC SSf Sister Val Thr val Thr ßly rro rro Ara ßlw rr »rhß ßlr Arq Lyß Kla SOS Jio 313 CTC AAO or ATT ßAT ere AM ATß AM AM ATO CCT ßAT CTC CCC ATO 1007 Law Lyß Ale Zle Aap Val A n Mat Aßn Lyo Mat Aro Val Wing Mee 320 223 330 ACT CTT OCT OTC ßTT OCC CTC TTT ßCC ßAT? ßC CCS AC GCC ATC MA 1055 Thr Law Ale Vßl Vßl Ale Law rhe Al * Aap ßlr rr Thr Ala Zla Ara 335 J4ß 545 350 CAC ere OCT TCC TOB MA OTA AM OM ACC CAO Aßß ATO CTT ees ATC us Aap Val Ala Ser Trp Ara Val Ly »ßlw Thr ßlw Arq Mat Val Ala Zla 3SS SCO 303 CSC ACC GM CTA ACC AM CTß ßßA OCA TCT 6TT CAS 6AA ßßß CCS ßAC 1151 Ara Thr ßlu Law Thr Lyo Lau ßly Wing Oor Val ßlu ßlu ßly rrß Aap 370 37S TM TOC ATC ATC ACC CCS CCS CAS AM CTC AAC 676 ACS ßCß ATC ßAC 11 '* Tyr Cyß Zlß He Thr rra rro ßlu Lyo Lau Aan Val Thr Wing Zla Aap SOS SSO 3fS MC TM CAC GAC CAC MC ATB CCC ATC OCC TTC TCC CTT CCC CCC TGT 1247 Thr Tyr? Sp Aa Ac¿ Ara Met Al * Mat Al a rhe Ser Law Ala Ala Cy * «0 < 0t 410 CCT CAC CTJC CCC CTC ACC ATC CSC CAC CCT ßßß TGC ACC V; AAC ACC 12 »* Wing CV * "ro« to Thr TU Ara AS »rrß ßl and Cy« Thr Arj Ly »Thr us 1: 0 4¿S« JO TTC CCC SM TTG? T GTG CT? ACC ACT TTC CTC AM AAT IJJ7 fh * fro Aap Tyr fhe Aßp Val Leu S * r Thr rhe Val Lyß Aan «1S 440 TAA lS40 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 444 amino acids (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Wing Cly Ale ßlw ßlw llß Val Leu ßln rro Zlß Ly »ßlu Zlß time Cly 1 S 10 13 Thr Val Lyo Law rro Cly Ser Lyß Sar Lau Ser Aan Arq Zlß Lau Lau 20 23 30 Leu Ala Ala Law Ser ßlw Cly Thr Thr Val Val Aap Aßn Lau Lau Aßn 35 40 45 Ser ßlu Aap Val NI * Tyr Met Law ßlr Al * Leu Arq Thr Lau Cly Leu 50 SS «0 * t Val Glu Ala Aa» Lyo Al * Al * Ly * Ara Al * Val Val Val Gly Cyß • S 70 71 SO ßly ßly Lyß fh * rro val ßlw Aap Al * Ly * ßlw ßlw Val ßln Lau rha OS M »S Lau ßly Aan Ala ßly II * Ala Met Ara% mt Law Thr Ala Ala Val Thr 100 101 110 Ala Ala ßly? A * it Thr Tyr Val Lew Aßp ßly Val rro Arq Mot 115 120 12S Ara ßlw Ara rro li * ßly Aßp Law val Val ßly Law Lyß ßln Lau Cly 130 11 »140 Ala Aap Val Aßp Cy * rhe Lew ßly Thr Aap Cyß rro rro Val Arq Val 141 ISO 1SS ICO Aßn Gly He ßly ßly Le Pro ßlr ßlr Lyo Vßl Lyß Leu Ser ßly Ser 1CS 170 175 Zlß Ser Ser Cln Tyr Law Ser Ala Law Lau Mßt? Ala Ala Lau Ala ISO IOS ISO ßly Aap Val ßlw lia ßlw Zla Zla Aap Ly * Leu? Lß Ser Zlßrro 1SS 200 205 Tyr al ßlu Mat Thr Law Ara Lau Mot ßlu Ara rho ßly Val Lv »Ale 210 ?? $ 220 ßlw Hlß Sar Aap Ser Trp Aap Ara P or Tyr Zlß Lvß ßr ßl ßln r» 225 230 235 240 Tyr Lyß Ala Tyr val ßlw ßly Aßp Ala ßßr Sor Wing 24 S 230 234 Ser Tyr rhß Lew Ala ßlr Ala Ala Zla Thr ßlr ßl Thr Val Thr Val 200 2CS 270 ßlw ßlr Cyß ßlr Thr Thr Ser Lau ßln ßlr Aßp Val Lyo Pho Ala ßlu 27S 200 205 val Lau Clu Met. r Cly Ala Ly »Val Thr Trp Thr Clu Thr Sor at 2 * 0 2 * \ 300 Thr vji Thr Cly Pra Pro Arg Clu Pro Pho Cly Arg Lys Hla Leu Lys JOS JIO JI * J: O Ato Il «Aso Val Aßh Mat Aon Ly» Met Pro Aßp Val Ala Mae Thr Lau 32 »JJ0 JJ * Ala vai to AI * Lau Pho Ala Aap Cly Pra Thr Ala Zl »? Rg Asp val J40 343 SSO Wing Ser Trp Arg vl Xy» Clu Thr Glu? Rg Hot Val? The? Lo? Rg Thr 333 3C0 JßS ßlu Lau Thr Ly »Lam Cly? Sister Val Clu Glu Cly Pre? ßp Tyr Cyß 970 37S 300 River Zlß Thr Pro Pro Clu Lyß Lau Aan Val Thx? La II»? Jrp Thr Tyr 305 3 * 0 393 400 ? sp Aop Hlß Arg Hot Ala Mßt? the Phß S r Leu Ala? the Cy *? la Clu 40S 410 41S Val Pro val Thr? L *? Rg Aßp Pro Gly Cy »Thr Arg Ly» Thr Phß Pro 420 423 430 Aap Tyr Phß Aap Val LOAI Ser Thr Fhe Val Lyßan It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (18)

1. CLAIMS 1. A DNA gene that encodes a mutated 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), characterized in that it comprises at least one threonine substitution 102 - > isoleucine
2. 2. DNA gene according to claim 1, characterized in that it also comprises at least one second mutation in the EPSPS, different from the first mutation.
3. 3. DNA gene according to claim 2, characterized in that it also comprises a mutation consisting of a substitution of proline 106 by serine.
4. 4. DNA gene according to claim 2, characterized in that it also comprises a mutation consisting of a substitution of glycine 101 for alanine.
5. 5. DNA gene according to any of claims 1 to 4, characterized in that it is of bacterial origin.
6. 6. DNA gene according to claim 5, characterized in that it originates from a bacterium of the genus Salmonella typhimurium.
7. 7. DNA gene according to any of claims 1 to 4, characterized in that it is of plant origin.
8. 8. DNA gene according to claim 7, characterized in that it is of maize origin.
9. 9. Protein of mutated EPSPS, characterized in that it comprises at least one substitution of threonine 102 for isoleucine.
10. 10. Chimeric gene comprising a coding sequence, as well as regulatory elements at the 5 'and 3' positions, which are heterologous and capable of functioning in plants, characterized in that it comprises, as coding sequence, at least one sequence in accordance with any of claims 1 to 8.
11. 11. Chimeric gene according to the claim 9, characterized in that it comprises a plant virus promoter.
12. 12. Chimeric gene in accordance with the claim 10, characterized in that it comprises a plant promoter (for example α-tubulin, histone, introns, actin, etc.).
13. 13. Vector for plant transformation, characterized in that it comprises at least one gene according to any of claims 10 to 12.
14. 14. Plant cell, characterized in that it comprises at least one gene according to any of claims 10 to 12.
15. 15. Plant, characterized in that it is obtained by regeneration of a cell according to claim 14.
16. 16. Method for the production of plants with improved tolerance to a herbicide having the EPSPS synthase as its target, characterized in that cells of plants or protoplasts are transformed with a gene according to any one of claims 1 to 8, and in that the transformed cells are subjected to regeneration.
17. 17. Method of treating plants with a herbicide having EPSPS as its objective, characterized in that the herbicide is applied to plants according to claim 15.
18. 18. Method according to claim 17, characterized in that glyphosate or a precursor is applied. of glyphosate.