AP886A - Mutated 5-enol pyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene. - Google Patents

Abstract

A mutated glyphosale resistance gene of 5-enol pyruvylshikimate-3-phosphate synthasc (EPSPS) including at least one substitution of threonmc 102 by isoleucine. and useful for producing glyphosate-resiscant transformed plants, is disclosed.

Description

Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for this protein and transformed plants containing this gene

The present invention relates to a new 5 5-enolpyruvylshikimate-3-phosphate synthase (or EPSPS) which displays increased tolerance with respect to herbicides which are competitive inhibitors with respect to phosphonoenolpyruvate (PEP) of EPSPS activity. This more tolerant EPSP synthase possesses at least one threonine by isoleucine substitution. The invention also relates to a gene coding for such a protein, to plant cells transformed by chimeric gene constructions containing this gene, to the plants regenerated from these cells and also to the plants originating from crossing using these transformed plants .

Glyphosate, sulfosate or fosametine are broad-spectrum systemic herbicides of the phosphonomethylglycine family. They act essentially as competitive inhibitors of 5-enolpyruvylshikimate-3phosphate 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 cauline and root apices, causing damage to the point of destruction of sensitive plants .

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Plastid EPSPS, the main target of these products, is an enzyme of the pathway of biosynthesis 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 plastids where it accumulates in its mature form.

The tolerance of plants to glyphosate and to products of the family is obtained by stable introduction into their genome of an EPSPS gene, of

-i. 10 plant or bacterial origin, which is mutated or

V. « otherwise in respect of the characteristics of inhibition by 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 which are used, it is advantageous to be able to express the product of the translation of this gene so as enable it to be accumulated in substantial amounts ·<>

in the plastids.

v-y It is known, for example from US Patent

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 genome of plants a gene coding for an EPSPS carrying at least one mutation that makes this enzyme more resistant to its competitive inhibitor (glyphosate) after localization of the enzyme in the plastid compartment. These techniques, however, need to be improved in order to obtain greater reliability in the use of these plants under agricultural conditions.

U υ ύ 8 8 6

-3Ιη the present description, plant is understood to mean any differentiated multicellular organism capable of photosynthesis, and plant cell is understood to mean any cell originating from a plant and capable of constituting undifferentiated tissues such as calluses or differentiated tissues such a embryos or plant parts or seeds.

The subject of the present invention is the production of transformed plants having increased tolerance to herbicides having EPSP synthase as their target, e.g. of the phosphonomethylglycine family, e.g. glyphosate or a glyphosate precursor, by regeneration of cells transformed by means of new chimeric genes containing a gene for tolerance to these herbicides.

The invention accordingly provides a DNA sequence coding for a mutated

5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), characterized in that the EPSPS is from maize and comprises a first mutation consisting in the threonine 102 -+ isoleucine substitution and a second mutation consisting in a substitution of proline 106 by serine; the invention also provides a DNA sequence coding for a mutated 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), characterised in that it comprises the coding region of the DNA sequence represented in SEQ ID No. 2, comprising a first mutation consisting in the threonine 102 isoleucine substitution and a second mutation consisting in a substitution of proline 106 by serine; and a DNA sequence coding for a mutated 5-enolpyruvylshikimate -3-phosphate synthase (EPSPS), characterised in that it comprises the coding sequence of SEQ ID No. 4.

According to a further feature of the invention there is provided a 5enolpyruvylshikimate -3-phosphate synthase (EPSPS), characterized in that the EPSPS is from maize and comprises a first mutation consisting in the substitution of threonine 102 by isoleucine and a second mutation consisting in a substitution of proline 106 by serine.

The invention also provides a chimeric gene comprising a coding sequence as well as regulatory elements at position 5' and 3' which are heterologous and capable of functioning in plants, characterized in that it comprises as coding sequence at least one sequence according to the invention.

ΑΡ ο ο ο β 8 6

3a

The chimeric gene of the invention for conferring on plants increased tolerance with respect to a herbicide having EPSPS as its target, generally comprises, in the direction of transcription: a promoter region, optionally a transit peptide region, a sequence of a gene coding for a glyphosate tolerance enzyme and an untranslated 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 threonine 102 by isoleucine substitution in the aroA (EPSPS) region and, in the same region, a proline 106 by serine substitution. These substitutions can be introduced or be present in an EPSPS sequence of any

AP Ο Ο Ο 8 8 6 origin, in particular of plant, bacterial, algal or fungal origin.

The transit peptides which can be used in the transit peptide region can be, known per se, of plant origin, for example originating from maize, sunflower, pea, tobacco or the like. The first and the second transit peptide can be identical, similar or different. They can, 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 permit the release of a mature and native protein, and especially the above mutated EPSPS, with maximum efficacy in the plasmid compartment.

The promoter region of the chimeric gene according to the invention may comprise a plant virus promoter; the region may be advantageously composed of at least one gene promoter or promoter fragment which is expressed naturally in plants (e.g. tubulin, generally α-tubulin, introns actin, histone).

The untranslated transcription termination signal region at the 3’ end of the chimeric gene may 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 Arabidopsis thaliana histone H4A748 gene according to the European Patent Application (European Application 633 317).

The chimeric gene according to the invention can comprise, in addition to the essential portions

AP ϋ υ Ο 8 8 6

-5above, at least one untranslated intermediate (linker) region, which can 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.

The method of the invention for the production of plants with improved tolerance to a herbicide having EPSP synthase activity as its target, characterised in that plant cells or protoplasts are transformed with a gene according to the invention and in that the transformed cells are subjected to a regeneration. The plants obtained may be used to prepare hybrid lines.

Isolation of a cDNA coding for a maize EPSPS:

The different steps which led to the obtaining of maize EPSPS cDNA, which served as substrate for the introduction of the two mutations, are described below. All the operations described below are given by way of example, and correspond to a choice made from among the different methods available for arriving at the same result. This choice has no effect on the quality of the result, and consequently any suitable method may be used by a person skilled in the art to arrive at the same result. Most of the methods of engineering of DNA fragments are described in Current Protocols in Molecular Biology Volumes 1 and 2, Ausubel ♦ F.M. et al., published by Greene Publishing Associates and Wiley-Interscience (1989) (hereinafter, references to protocols described in this work will be designated ref. CPMB). The operations relating to DNA which were performed according to the protocols described in this work are especially the following: ligation of DNA fragments, treatment with Klenow DNA polymerase and T4 DNA polymerase, preparation of plasmid and of bacteriophage λ DNA, either as a minipreparation or as a maxipreparation, and DNA and

AP υ υ ϋ 8 β 6 minipreparation or as a maxipreparation, and DNA and RNA analyses according to the 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 of an Arabidopsis thaliana EPSPS fragment

a) Two 20-mer oligonucleotides of respective sequences :

5'-GCTCTGCTCATGTCTGCTCC-3'

5’-GCCCGCCCTTGACAAAGAAA-3’ were synthesized from the sequence of an Arabidopsis thaliana EPSPS gene (Klee H.J. et al. (1987) Mol. Gen. Genet., 210, 437-442). Tksse two oligonucleotides are at positions 1523 to 1543 and 1737 to 1717, respectively, of the published sequence, and in opposite orientations.

b) Arabidopsis thaliana (var. Columbia) total

DNA was obtained from Clontech (catalogue reference:

6970-1).

c) 50 nanograms (ng) of DNA are mixed with 300 ng of each of the oligonucleotides and subjected to

35 amplification cycles with a Perkin-Elmer 9500 apparatus, under the conditions of standard medium for amplification which are recommended by the supplier.

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The resulting 204-bp fragment constitutes the

Arabidopsis thaliana EPSPS fragment.

2. Construction of a library of a cDNA from a

BMS maize cell line

a) 5 g of filtered cells are ground in liquid nitrogen, and the total nucleic acids are extracted according to the method described by Shure et al. with the following modifications:

the pH of the lysis buffer is adjusted to PH 9.0;

after precipitation with isopropanol, the pellet is taken up in water and, after dissolution, adjusted to 2.5 M

LiCl. After incubation for 12 h at °C, the pellet from centrifugation for 15 min at 30,000 g at 4°C is resolubilized. _r The LiCl precipitation step is then repeated. The resolubilized pellet constitutes the RNA fraction of the total nucleic acids.

b) The poly(A)⁺ RNA fraction of the RNA fraction is obtained by chromatography on an oligo(dT)cellulose column as described in Current Protocols in

Molecular Biology.

c) Synthesis of double-stranded cDNA having a synthetic EcoRI end: this is carried out according to the protocol of the supplier of the different reagents needed for this synthesis penny form of a kit: the

AP/P/ 98/01 195

AP 0 U Ο 8 8 6 copy kit from the company In Vitrogen.

Two single-stranded and partially complement airy oligonucleotides of respective sequences:

5'-AATTCCCGGG-3'

5'-CCCGGG-3' (the latter being phosphorylated) are ligated with the blunt-ended double-stranded cDNAs.

This ligation of the adaptors results in the creation of Smal sites attached to the double-stranded cDNAs and EcoRI sites in cohesive form at each end of the double-stranded CDNAs.

d) Creation of the library:

The cDNAs possessing the artificial cohesive

EcoRI sites at their ends are ligated with bacteriophage XgtlO cDNA which has 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, namely Gigapack Gold, according to the supplier's instructions; this library was titrated using the bacterium E. coli C600hfl. The library thereby obtained is amplified and stored according to the instructions of the same supplier, and constitutes the BMS maize cell suspension cDNA library.

3. Screening of the BMS maize cell suspension cDNA library with the AraJbidopsis thaliana EPSP probe The protocol followed is that of Current

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ΑΡ ο ο Ο 8 8 6

Protocols in Molecular Biology Volumes 1 and 2,

Ausubel F.M. et al., published by Greene Publishing Associates and Wiley-Interscience (1989)(CPMB).

Briefly, approximately 10⁶ recombinant phages are plated out on LB dishes at an average density of 100 phages/cm². The lytic plaques are replicated in duplicate on Amersham Hybond N membranes.

h) The DNA was fixed to the filters by 1600kJ

UV treatment (Stratagene Stratalinker). The filters were prehybridized in 6xSSC/0.1%SDS/O.25 skimmed milk for 2 h at 65°C. The Arabidopsis thaliana EPSPS probe was labelled with [³²P]dCTP by random priming according to the supplier's instructions (Pharmacia Ready to Go kit). The specific activity obtained is of the order of

10⁸ cpm per μg of fragment. After denaturation for 5 min at 100°C, the probe is added to the prehybridization medium and hybridization is continued for 14 hours at 55°C. The filters are fluorographed for h at -80°C with Kodak XAR5 film and Amersham

Hyperscreen RPN enhancing screens. Alignment of the positive spots on the filter with the dishes from which, they originate enables zones corresponding to the phages displaying a positive hybridization response with the Arabidopsis thaliana EPSPS probe to be picked out from the dish. This step of plating out, transfer, hybridization and recovery is repeated until all the spots in the dish of the successively purified phages prove 100¾ positive in hybridization. A plaque of

98/01195 i‘.: ··«»

ΑΡ υ ο ΰ 8 8 6 independent phage lysis is then picked out in diluent λ medium (Tris-Cl pH 7.5; lOmM MgSO₄; 0.1M NaCl; 0.1¾ gelatin); these phages in solution constitute the EPSPpositive clones of the BMS maize cell suspension.

4. Preparation and analysis of the DNA of the

EPSP clones of the BMS maize cell suspension

Approximately 5xl0⁸ phages are added to 20 ml of C600hfl bacteria at an OD_600nm value of 2/ml and incubated for 15 minutes at 37°C. This suspension is then diluted in 200 ml of bacterial growth medium in a

1-1 Erlenmeyer and stirred in a rotary stirrer at 250 rpm. Lysis is noted when the medium clarifies, corresponding to the lysis of the turbid bacteria, and takes place after approximately 4 h of stirring. This supernatant is then treated as described in Current Protocols in Molecular Biology. The DNA obtained corresponds to the EPSP clones of the BMS ma4,ze cell suspension.

One to two gg of this DNA are cut with EcoRI and separated on 0.8¾ LGTA/TBE agarose gel (ref. CPMB). A final verification consists in checking that the purified DNA does indeed display a hybridization signal with the Arabidopsis thaliana EPSPS probe. After electrophoresis, the DNA fragments are transferred onto

Amersham Hybond N membranes according to the protocol of Southern described in Current Protocols in

Molecular Biology. The filter is hybridized with the

Arabidopsis thaliana EPSPS probe according to the

Ci o

oo a

AP U Ο ϋ 8 8 6 conditions described in section 3 above. The clone displaying a hybridization signal with the Arabidopsis thaliana EPSPS probe and containing the longest EcoRI fragment has a size estimated on gel as approximately

1.7 kbp.

5. Obtaining of the clone pRPA-KL-711 Ten qg of the phage clone containing the

1.7-kbp insert are digested with EcoRI and separated on 0.8% LGTA/TBE agarose gel (ref. CPMB). The gel fragment containing the 1.7-kbp insert is excised from the gel by BET staining, and the fragment is treated with β-agarse according to the protocol of the supplier, New

CD

the 1.7-kbp	O>
with the DNA of
	OO
cut with EcoRI	o

0« according to the ligation protocol described in

Current Protocols in Molecular Biology. Two μΐ of the above ligation mixture are used for the transformation of an aliquot of electrocompetent E. coli DH10B; transformation is accomplished by electroporation using the following conditions: the mixture of competent bacteria and and ligation medium is introduced into an electroporation cell of thickness 0.2 cm (Biorad) previously cooled to 0°C. The physical conditions of the electroporation using an electroporator made by Biorad are 2500 volts, 25 μΕ and 200 Ω. Under these conditions, the mean discharge time of the condenser is of the order of 4.2 milliseconds. The bacteria are then

Al υ „ ύ 8 8 6 taken up in 1 ml of SOC medium (ref. CPMB) and stirred for 1 hour at 200 rpm on a rotary stirrer in 15-ml Corning tubes. After plating out on LB/agar medium supplemented with 100 Bg/ml of carbenicillin, minipre5 parations of the bacterial clones which have grown after one night at 37°C is produced according to the protocol described in Current Protocols in Molecular Biology. After digestion of the DNA with EcoRI and separation by electrophoresis on 0.8¾ LGTA/TBE agarose gel (ref. CPMB), the clones possessing a 1.7-kbp insert are retained. A final verification consists in checking that the purified DNA does indeed display a hybridization signal with the Arabidopsis thaliana EPSPS probe. After electrophoresis, the DNA fragments are transferred onto Amersham Hybond N membranes according to the protocol of Southern described in Current Protocols in Molecular Biology. The filter is hybridized with the Arabidopsis thaliana EPSPS probe according to the conditions described in section 3 above. The plasmid clone possessing a 1.7-kbp insert and hybridizing with the Arabidopsis thaliana EPSPS probe was prepared on a larger scale, and the DNA resulting from the lysis of the bacteria was purified on a CsCl gradient as described in Current Protocols in Molecular Biology. The purified DNA was partially sequenced with a Pharmacia kit according to the supplier's instructions and using as primers the M13 direct and reverse universal primers ordered from the in c>

co

CP

A

At

AP Ο Ο Ο 8 8 6 same supplier. The partial sequence produced covers approximately 0.5 kbp. The derived amino acid sequence in the region of the mature protein (approximately 50 amino acid residues) displays 100¾ identity with the corresponding amino sequence of mature maize EPSPS described in American Patent USP 4,971,908). This clone, corresponding to a 1.7-kbp EcoRI fragment of the EPSP DNA of the BMS maize cell suspension, was designated pRPA-ML-711. The complete sequence of this clone was determined on 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 of this 1713-bp clone is presented in SEQ ID

No. 1.

6. Obtaining of the clone pRPA-ML-715

Analysis of the sequence of the clone pRPAML-711, and especially comparison of the derived amino acid sequence with that of maize, shows a sequence 20 extension of 92 bp upstream of the GCG codon coding for the NH₂-terminal alanine of the mature portion of maize EPSPS (American Patent USP 4,971,908). Similarly, an extension of 288 bp downstream of the AAT codon coding for the COOH-terminal asparagine of the mature portion of maize EPSPS (American Patent USP 4,971,908) is observed. These two portions could correspond, in the case of the NH₂-terminal extension to a portion of the sequence of a transit peptide for plastid localization,

P/P/ 9 8/01195

APO00886 and, in the case of the COOH-terminal extension, to the untranslated 3' region of the cDNA.

In order to obtain a cDNA coding for the mature portion of the maize EPSPS cDNA, as described in 5 USP 4,971,908, the following operations were carried out:

a) Removal of the untranslated 3' region:

construction of pRPA-ML-712:

The clone pRPA-ML-711 was cut with the restriction enzyme Asel, and the ends resulting from this cleavage were rendered blunt by treatment with the

Klenow fragment of DNA polymerase I according to the protocol described in CPMB. A cleavage with the restriction enzyme SacII was then performed. The DNA 15 resulting from these operations was separated by electrophoresis on 1¾ LGTA/TBE agarose gel (ref. CPMB).

The gel fragment containing the 0.4-kbp ’ £'b,

Asel-blunt ends/SacII insert was excised 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 Hindlll [lacuna] located in the polylinker of the cloning vector pUC19, and the ends resulting from this cleavage were rendered blunt by treatment with the Kl-now fragment of DNA polymerase I. A cleavage with the restriction enzyme

SacII was then performed. The DNA resulting from these manipulations was separated by electrophoresis on 0.7¾

LGTA/TBE agarose gel (ref. CPMB).

8/01 195

ΑΡ υ υ υ 8 a β

The gel fragment containing the approximately

3.7-kbp Hindlll-blunt ends/SacII insert was excised from the gel and purified according to the protocol described in section 5 above.

The two inserts were ligated, and 2 μΐ of the ligation mixture were used to transform E. coli DH10B as described above in section 5.

The plasmid DNA content of different clones was analysed according to the procedure described for pRPA-ML-711. One of the plasmid clones selected contains an approximately 1.45-kbp EcoRI-Hindlll insert. The sequence of the terminal ends of this clone reveals that the 5' end of the insert corresponds exactly to the corresponding end of pRPA-ML-711, and that the 3’-terminal end possesses the following sequence:

5'- . . .AATTAAGCTCTAGAGTCGACCTGCAGGCATGCAAGCTT-3'

8/01 195

The underlined sequence corresponds to the codon of the COOH-terminal amino acid asparagine, the 20 next codon corresponding to the translation stop codon.

The nucleotides downstream correspond to sequence elements of the pUC19 polylinker. This clone comprising the pRPA-ML-711 sequence up to the translation termination site of mature maize EPSPS and followed by 25 sequences of the pUC 19 polylinker up to the Hindlll site was designated pRPA-ML-712.

b) Modification of the 5' end of pRPA-ML-712:

construction of pRPA-ML-715:

AP v ϋ Ο 8 8 6

The clone pRPA-ML-712 was cut with the restriction enzymes Pstl and Hindlll. The DNA resulting from these manipulations was separated byelectrophoresis on 0.8¾ LGTA/TBE agarose gel (ref.

CPMB). The gel fragment containing the 1.3-kbp PstlEcoRI insert was excised 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 10 complementary- oligonucleotides of sequence:

Oligo 1: 5'-GAGCCGAGCTCCATGGCCGGCGCCGAGGAGATCGTGCTGCA-3'

Oligo 2: 5'-GCACGATCTCCTCGGCGCCGGCCATGGAGCTCGGCTC-3' as well as in the presence of plasmid pUC19 DNA digested with the restriction enzymes BamKT and

Hindlll.

Two μΐ of the ligation mixture were used to transform E. coli DH10B as described above in section £·Γ <r

5. After analysis of the plasmid DNA content of different clones according to the procedure described above in section 5, one of the clones possessing an approximately 1.3-kbp insert was retained for subsequent analyses. The sequence of the 5'-terminal end of the selected clone reveals that the DNA sequence in this region is the following: sequence of the pUCl9 polylinker from the EcoRI to the BamHI sites, followed by the sequence of the oligonucleotides used in the cloning, follow^Ted by the remainder of the sequence present in pRPA-ML-712. This clone was designated pRPASGI I 0 / 8 6 /d

APO00886

ML-713. This clone possesses a methionine ATG codon included in an Ncol site upstream of the N-terminal alanine codon of mature EPSP synthase. Furthermore, the alanine and glycine codons of the N-terminal end have been preserved, but modified on the third variable base: initial GCGGGT gives modified GCCGGC.

The clone pRPA-ML-713 was cut with the restriction enzyme Hindlll, and the ends of this cleavage were rendered blunt by treatment with the

Klenow fragment of DNA polymerase I. A cleavage with restriction enzyme Sacl was then performed. The DNA resulting from these manipulations was separated by electrophoresis on 0.8¾ LGTA/TBE agarose gel (ref. CPMB). The gel fragment containing the 1.3-kbp

Hindlll-blunt ends/SacI insert was excised 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 restriction enzyme Xbal, and the ends of this cleavage were rendered blunt by treatment with the Klenow fragment of

DNA polymerase I. A cleavage with the restriction enzyme Sacl was then performed. Two μΐ of the ligation mixture were used to transform E. coli DH10B as described above in section 5. After analysis of the plasmid DNA content of different clones according to the procedure described above in section 5, one of the clones possessing an approximately 1.3-kbp insert was retained for subsequent analyses. The sequence of the

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ΑΡϋ 0 0 8 8 6 terminal ends of the selected clone reveals that the

DNA sequence is the following: sequence of the pUCl9 polylinker from the EcoRI to Sacl sites, followed by the sequence of the oligonucleotides used in the cloning from which the 4 bp GATCC of the oligonucleotide 1 described above have been deleted, followed by the remainder of the sequence present in pRPA-ML-712 up to the Hindlll site and sequence of the pUC19 polylinker from Xbal to Hindlll. This clone was designated pRPA-ML-715.

7. Obtaining of a cDNA coding for a mutated maize EPSPS

All the mutagenesis steps were carried out with the Pharmacia U.S.E. mutagenesis kit according to the supplier's instructions. The principle of this mutagenesis system is as follows: plasmid DNA is denatured by heat and reassociated in the presence of a molar excess of, on the one hand the mutagenesis oligonucleotide, and on the other hand an oligonucleotide enabling a unique restriction enzyme site present in the polylinker to be eliminated. After the reassociation step, synthesis of the complementary strand is carried out by the action of T4 DNA polymerase in the presence of T4 DNA ligase and gene 32 protein in a suitable buffer which is supplied. The synthesis product is incubated in the presence of the restriction enzyme for which the site is assumed to have disappeared by mutagenesis. The E. coli strain

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ΑΓ 000886 possessing, in particular, the xnutS mutation is used as host for the transformation of this DNA. After growth in liquid medium, the total plasmid DNA is prepared and incubated in the presence of the restriction enzyme used before. After these treatments, E. coli strain

DH10B is used as host for the transformation. The plasmid DNA of the clones isolated is prepared, and the presence of the mutation introduced is verified by sequencing.

A)- modification of sites or sequences without in principle affecting the EPSPS-resistance character of maize to products which are competitive inhibitors of EPSP synthase activity: elimination of an internal Ncol site from pRPA-ML-715.

The pRPA-ML-715 sequence is numbered arbitrarily by placing the first base of the N-terminal alanine codon GCC at position 1. This sequence possesses an Ncol site at position 1217. The sitemodification oligonucleotide possesses the sequence:

5 '-CCACAGGATGGCGATGGCCTTCTCC-3'.

After sequencing according to the references given above, the sequence read after mutagenesis corresponds to that of the oligonucleotide used. The Ncol site has indeed been eliminated, and the 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

AP/P/ 9 8/01 195

ΑΡ κ ο ο 8 8 β presented in SEQ ID No. 2 and SEQ ID No. 3.

B)- sequence modifications enabling the

EPSPS-resistan.ee character of maize to products which are competitive inhibitors of EPSP synthase activity to be increased.

The following oligonucleotides were used:

a) mutation Thr 102 —> lie.

5'-GAATGCTGGAATCGCAATGCGGCCATTGACAGC-3'

b) mutation Pro 106 -+ Ser.

5 ' -GAATGCTGGAACTGCAATGCGGTCCTTGACAGC-3 ·

c) mutations Gly 101 -+ Ala and Thr 102 —> lie.

5'-CTTGGGGAATGCTGCCATCGCAATGCGGCCATTG-3'

d) mutations Thr 102 -+ lie and Pro 106 —> Ser.

5'-GGGGAATGCTGGAATCGCAATGCGGTCCTTGACAGC-3'

After sequencing, the sequence read after mutagenesis on the three mutated fragments is identical to the parent pRPA-ML-716 DNA sequence, with the exception of the mutagenized region which corresponds to that of the mutagenesis oligonucleotides used. These clones were designated: pRPA-ML-717 for the mutation

Thr 102 -+ lie, pRPA-ML-718 for the mutation Pro 106 -+

Ser, pRPA-ML-719 for the mutations Gly 101 —> Ala and

Thr 102 -+ lie and pRPA-ML-720 for the mutations Thr 102

-+ lie and Pro 106 -+ Ser.

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AP V Ο 0 8 8 6

The 1340-bp sequence of pRPA-ML-720 is presented in SEQ ID No. 4 and SEQ ID No. 5.

The 1395-bp Ncol-Hindlll insert is the basis of all the constructions used for the transformation of plants for the introduction of resistance to herbicides which are competitive inhibitors of EPSPS, and especially glyphosate resistance. This insert will be designated in the remainder of the description the maize EPSPS double mutant.

Example 2;

Glyphosate tolerance of the different mutants in vitro

2.a: Extraction of EPSP synthase The different EPSP synthase genes are introduced in the form of an Ncol-Hindlll cassette into the plasmid vector pTrc99a (Pharmacia, ref: 27-5007-01) cut with Ncol and Hindlll. Recombinant E. coli DH10B * bacteria overexpressing the different EPSP synthases are sonicated in 40 ml of buffer per 10 g of pelleted cells, and washed with this same buffer (200 mM TrisHC1 pH 7.8, 50 mM mercaptoethanol, 5 mM EDTA and 1 mM PMSF), to which 1 g of polyvinylpyrrolidone is added. The suspension is stirred for 15 minutes at 4°C and then centrifuged for 20 minutes at 27,000 g and 4°C.

Ammonium sulphate is added to the supernatant to bring the solution to 40% saturation with respect to ammonium sulphate. The mixture is centrifuged for 20 minutes at 27,000 g and 4°C. Ammonium sulphate is

7P/ 9 8/01 195

APO 0 0 8 8 6 added to the new supernatant to bring the solution to

70¾ saturation with respect to ammonium sulphate. The mixture is centrifuged for 30 minutes at 27,000 g and

4°C. The EPSP synthase present in this protein pellet is taken up in 1 ml of buffer (20 mM Tris-HCl pH 7.8 and 50 mM mercaptoethanol). This solution is dialysed overnight against two litres of this same buffer at

4°C.

2.b: Enzyme activity

The activity of each enzyme, as well as its glyphosate resistance, is measured in vitro over 10 minutes at 37°C in the following reaction mixture:

100 mM maleic acid pH 5.6, 1 mM phosphoenolpyruvate, mM shikimate 3-phosphate (prepared according to

Knowles P.F. and Sprinson D.B. 1970. Methods in Enzymol 17A, 351-352 from Aerobacter aerogenes strain ATCC 25597) and 10 mM potassium fluoride. The enzyme extract is added at the last moment after the addition of glyphosate, the final concentration of which varies from 0 to 20 mM.

The activity is measured by assaying the phosphate liberated according to the technique of Tausky H.A. and Shorr E. 1953. J. Biol. Chem. 202, 675685.

Under these conditions, the wild-type (WT) enzyme is already 85¾ inhibited at a glyphosate concentration of 0.12 mM. At this concentration, the mutant enzyme known as Serl06 is only 50¾ inhibited,

AP/P/ 9 8/01 195

V*

ΑΡ υ u o 8 8 6 and the other three mutants, Ilel02, Ilel02/Serl06 and

Alal01/Ilel02, show little or no inhibition.

The glyphosate concentration has to be multiplied by ten, that is to say 1.2 mM, in order to 5 produce a 50¾ inhibition of the mutant enzyme Ilel02, the mutants Ilel02/Serl06, Ala/Ile and Ala still not being inhibited.

It should be noted that the activity of the mutants Ala/Ile and Ala is not inhibited up to glyphosate concentrations of lOmM, and that that of the mutant Ilel02/SerlO6 is not reduced even if the glyphosate concentration is multiplied by 2, that is to say 20 mM.

Example 3;

Resistance of transformed tobacco plants

1-1- Transformation

The vector pRPA-RD-173 is introduced into

Ag-robacterium tumefaciens strain EHA101 (Hood et al.,

1987) carrying the cosmid pTVK291 (Komari et al.,

1986). The transformation technique is based on the procedure of Horsh et al. (1985).

1-2- Regeneration

The regeneration of PBD6 tobacco (source SEITA France) from leaf explants is carried out on a

Murashige and Skoog (MS) basal medium comprising 30 g/1 of sucrose as well as 200 pg/ml of kanamycin. The leaf explants are removed from plants cultivated in the greenhouse or in vitro and transformed according to the in σ>

o

A

Ar U J 0 8 8 6 leaf disc technique (Science, 1985, Vol. 227, pp. 12291231) in three successive steps: 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 (NAA) and 2 mg/1 of benzylaminopurine (BAP) for 15 days. The shoots formed during this step are then developed for 10 days by culturing on an MS medium supplemented with 30 g/1 of sucrose but not containing any hormone. Shoots which have developed are then removed and cultured on an MS rooting medium having half the content of salts, vitamins and sugar and not containing any hormone.

After approximately 15 days, the rooted shoots are transferred to soil.

1-3- Glyphosate resistance

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 indices recorded 3 weeks after treatment. Under these conditions, it is found that the plants transformed with the construction pRPA-RD-173 display very good tolerance, whereas the untransformed control plants are completely destroyed.

These results show clearly the improvement

AP/P/ 9 8/01 195

AP ν Ο ϋ 8 8 6 brought about by the use of a chimeric gene according to the invention for the same gene coding for glyphosate tolerance.

Example 4;

Transformation and selection of maize cells

BMS (Black Mexican Sweet) maize cells in an exponential growth phase are bombarded with the construction 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 pp. 70-73).

Two days after bombardment, the cells are transferred to the same medium containing 2 mM

N-(phosphomethyl) glycine.

After 8 weeks of selection on this medium, calluses which develop are selected, then amplified and <* ; analysed by PCR, and reveal clearly the presence of the chimeric OTP-EPSPS gene.

Cells not bombarded and grown on the same medium containing 2 mM N-(phosphomethyl)glycine are blocked by the herbicide and do not develop.

The transformed plants according to the invention may be used as parents for obtaining lines and hybrids having the phenotypic character corresponding to the expression of the chimeric gene introduced.

AP/P/ 9 8/01 195

AP U ΰ ϋ 8 8 6

Description of the constructions of the plasmids pRPA-RD-124: Addition of a nos polyadenylation signal to pRPA-ML-720 with creation of a cloning cassette containing the maize double mutant EPSPS gene (Thr 102 —> lie and Pro 10 6-4 Ser). pRPA-ML720 is digested with Hindlll and treated with the Klenow fragment of E. coli DNA polymerase I to produce a blunt end. A second digestion is performed with Ncol, and the EPSPS fragment is purified. The EPSPS gene is 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 vector pRPA-RD-12, it was necessary for the latter to be digested beforehand with Sail, 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) is digested with SphI, treated with T4 DNA polymerase and then digested with Spel, and the OTP fragment is purified. This OTP fragment is cloned into pRPA-RD-124 which has previously been digested with

Ncol, treated with Klenow DNA polymerase to remove the protruding 3' portion and then digested with Spel. This clone is then sequenced in order to ensure correct

AP/P/ 9 8/01195

AP 0 U ϋ 8 8 6 translational fusion between the OTP and the EPSPS gene. pRPA-RD-125 is then obtained.

pRPA-RD-130: Addition of the H3C4 maize histone promoter and of adhl intron 1 sequences of 5 pRPA-RD-123 (Patent Application EP 507 698) to pRPA-RD125 with creation of a cassette for expression in plants for the expression of the double mutant EPSPS gene in the tissues of monocotyledons. pRPA-RD-123 (a cassette containing the H3C4 maize histone promoter fused with the adhl intron 1) is digested with Ncol and Sad. The DNA fragment containing the promoter derived from pRPA-RD-123 is then purified and ligated with pRPA-RD-125 which has previously been digested with

Ncol and Sacl.

pRPA-RD-159: Addition of the H4A748

Arabidopsis histone double promoter (Patent Application EP* 5 07 698) to pRPA-RD-125 with creation of a cassette for expression in plants for the expression of the OTP-double mutant EPSPS gene gene in the tissues of dicotyledons. pRPA-RD-132 (a cassette containing the

H4A748 double promoter (Patent Application EP 507 698)) is digested with Ncol and Sacl. The purified promoter fragment is then cloned into [lacuna] which has been digested with Ecol and Sacl.

pRPA-RD-173: Addition of the H4A748 promoter-OTP-double mutant EPSPS gene gene of pRPA-RD159 to plasmid pRPA-BL-150A (European Patent

Application 508 909) with creation of an Agrobacterium

AP/P/ 9 8/01 195

AP 0 0 0 8 8 6 tumefaciens transformation vector. pRPA-RD-159 is digested with Notl and treated with Klenow polymerase. This fragment is then cloned into pRPA-BL-150A with

Smal.

AP/P/ 9 8/01 195

AP u υ u 8 8 6 sequence t.tsTtNc;

(Il GKMKKAl. ΙΝΡΚΜΛΤΙΟΜ:

Λγ'Μ.Γ-'ΗΤ

Lctirun, Micnel IX.· RicnJrt T “j Iιland. Ala in

I 111 TITLE OF INVENTION: S-enoi pyruvyishlfcinate-J-pnoapdaLe jynr.naae rut··, qane coda nt pout cette procaine ac plane» crane to meea concanant ca gana

Illl) HUMBER OF SEQUENCES: 5 llv) CORRESPONDENCE ADDRESS:

IA) ADDRESSEE: Francois Qaretlen

IB) STREET: 1420 rue Pirra Bailee

IC) CITY: Lyon Cadax 0»

IE) COUNTRY: France

IF) IIP: 69263

Iv) COMPUTER READABXE FORM:

(A) MEDIUM TYPE: Floppy diet

IB) COMPUTER: IBM PC coapatibla

IC) OPERATING SYSTEM: PC-DOSZMS-DOS

ID) SCTO4ARE: Patantln Ralaaaa 11.0, Varalon >1.25 (vi) CURRENT APPLICATION DATA:

IA) APPLICATION NUMBER:

IB) FILING DATE:

IC) CLASSIFICATION:

tviill ATTORNEY/AGENT INFORMATION:

(A) NAME: Qnratlan, Francois lixl TELECCWUNICATION INFORMATION:

(A) TELEPHONE: (331 72-29-26-46 (B) TELEFAX: (33)72-29-28-43

121 INFORMATION FOR SEQ ID NO:1:

11) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1713 base pairs (BI TYPE: nucleic acid (C) STRANDEDNESS: double I DI TOPOLOGY: linear

111) MOLECULE TYPE: cDNA (vll ORIGINAL SOURCE:

' I A) ORGANISM: 2ea nay» (B) STRAIN: Blact Mexican Ev»·: (FI TISSUE TYPE: Callua (vii) D-MEDIATF SOURCE:

(A) LIBRARY: laetoda gtlO IB) CLONE: pRPA-ML-711

AP/P/ 9 8/01 195

Ixil SEQUENCE DESCRIPTION: SEQ ID NO:1:

AATCAATTTC ACACAGGAAA CAGCIATGAC CATGATTACG AATTO3GGCC CGGGCGCGTG 60

ATCCSGCGGC GGCAGCGGCG GCGGCGGTGC AGGCGGGTGC C3AG&AGATC GTGCTSCRGC 120

CCATCAAGGA GATCTCOGGC ACOGTCAAGC TGOOGGGGTC CAAGTCGCTT 1CCAMXSGA 180

TCCTCCIACT OGCCGCLL'IS TCCGAGGGGA CAACAGTGGT TGATAACCTG CTOAACAGTO 240

AGGATGTCCA CTACATGCTC GGGGCCTTGA CGACTOTGG TCTCTCTGTC GAAGCGGACA 300

AAGCTGCCAA AAGAGCTGTA GTTGTTGGCT GTGGTGGAAA GTTCCCAGTT GAGGATGCIA 360

AAGAGGAAGT GCAGCTCTTC TTGGGGAATG CTGGAACTGC AATGCGGCCA TTGACACCAG 420

CTGTTACTGC TGCTGGTGGA AATGCAACTI ACGTGCTTGA. TGSAGTACCA AGAATGAGGG 480 αι’Λ'μΧτ^λτ Yf4zri*crh. tTrrtrranfiAT wanznct mmantt gttgatyctt tcctt.-x-cac ttact.-sttca cctcttottg tcaatccaat ccgaoggcta cctcctcgca agctcaagct <τστ·-·ζτττ; κτζΗ',ζκττζ tmterTwe tgccttgctg atcgcccctc ctttggctct Tf-'^iATirrc gagattgaaa tcattgataa attaatctcc attccgtacg

TCSAAATCAC ATTGAIIA7T0 ATCGAGCCTT TTGGTOTCAA AGCAGA3CAT TCTCATAGCT OGGACAGATT CTACATTAAG GGAGGTCAAA AATACAAGTC CCCTAAAAAT GCCTATG7TG

AAGGTGATGC CTCAAGOGCA AGCTATTTCT TGGCTGGTGC TGCAATTACT GGAGCGAJCTG TGACTGTGGA AGGTTGTGGC ACCACCAGTT TGCAGGGTGA TGTGAAGTTT GCTGAGGTAC TW3AGATGA7 GGGAGCGAAG GTTACATGGA CCGAGACTAJG CGTAACTGTT ACTGGCOCAC CGCGGGAGCC ATTIGGGAGG AAACACCTCA AGGCGATTGA TGTCAACATG AACAAGATGC CTGATGTCGC CATGACTCTT GCTGTGGTTG CCCTCTTTGC CGATGGCOCG ACAGCCATCA

SAfiACGTGGC TTCCTGGAGA GTAAAGGAGA CCGAGAGGAT GGTTGCGATC CGGACGGMC TAAOCAAGCI GGGAGCATCT GTTGACGAAG GGCCGGACTA CTGCA3CATC ACGCCGCCGG

AGAAGCTGAA CGTGACSGCG ATOGACAOGT ACGACGAOCA CAGGATGGCC ATGGOCTTCT (XCTTGCOGC CTGTGCCGAG GTOCCCGTCA CCATCCGGGA CCCTGGCTGC ACCCGGAAGA ccttccccga ctacttcgat gtgctgagca ctttcgtcaa gaattaataa agcgtgcgat

ACTACCACSC AGCTTGATTG AACTGA1AGG CTTGTGCTGA GGAAATACAT TTCT7TTGTT ctgti 'mcr ctttcacggg attaagi ttt gagtctgtaa cgttagttgt ttgtagcaag

TTTCTATTTC GGATCT7AAG 7TTGTGCACT GTAAGCCAAA TTTCA7TTCA AGAGTGGTTC

GTTGGAATAA 7AAGAA7AAT AAAT7ACGTT TCXGTGAAAA AAAAAAAAAA AAAAAAAAAA

AAAAAAAAAA AAAAAAAAAA AACCCSGGAA TTC {21 INFORMATION FOR SEQ ID NO:2:

(11 SEQUENCE CHARACTERISTICS:

(Al LENGTH: 1340 beee pelr*

IB] TYPE: nucleic tcid (C) STRANDEDNESS: double (DI TOPOLOGY: linetr (11) MOLECULE TYPE: cDNA (Vi) ORIGINAL SOURCE:

(A) ORGANISM: let uyc

IB) STRAIN: BIect Mexican Sweet (vil) ΙΑΚΧΟ LATE SOURCE:

(BI CLONE: pRPA-«L-71« (lx) FEATURE:

(A) NAME/KEY: COS (BI LOCATION: «..1337

510 •100 bNO

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

I Ί3

VP/ 9 8/01 195 <ί (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

CCATG GOT GGC GCC GAG SAG ATC GTG CTG CAG CCC ATC AAG GAG ATC 47

Ala Gly Ale Clu Glu lie Vel Leu Gin Pro lie Lye Glu lie

10

TCC Ser IS

GGC ACC Gly Thr

CTC ΛΑ3

CTG COG GGG

TCC Ser

AAG Lye

tog err

TCC AAC CGG ATC

95

Val

Lye

Leu 20

Pre Gly

Ser 25

Leu

Ser Aen

Arg

lie 30

CTC

CTA

CTC

ccc

GGC

CTG

TCC

GAG

GGG

ACA

ACA.

GTG

GTT

GAT

AAC

CTG

143

Leu

Leu

Leu

Ala

Ala

Leu

Ser

Glu

Gly

Thr

Thr

Val

Vel

A*p

A*n

Leu

35

40

45

CTG

AAC

xrr

GW

GAT

CTC

CAC

TAG

XTG

CTC

GGG

GCC

TTG

AGG

ACT

err

191

Leu

A»n

Ser

Glu

Aep

Vel

Hl*

Tyr

Het

Leu

Gly

Ale

Leu

Arq

Thr

Leu

ΑΡϋϋ ΰ 8 8 6

SO 55 ήΟ

G.CT GL·/

CTC TCT

CTC GAA OCT GAC AAA GCT

GCC Ala

AAA Lyy

AGA GCT Arg Ala ;s

CTA CTT GTT

2J’

L«-u

Car h'l

y.it

Glu

Ala

Aup Lys Ala 70

7 a I

Val

Val

'>·>τ

TCT

OCT

CCA

AAG

TTC

CCA GTT GAG

GAT

GCT

AAA OAO

GAA

CTG

CAG

Xd7

Gly

Cys dO

Gly

liy

Lys

Phe

Pro Vel Glu 35

Arp

Ala

Lyu Glu 90

G lu

v.U

Gin

CTC

TTC

TTG

GSG

AAT

GCT

GGA ACT GCA

ATG

CGG

CCA TTG

ACA

GCA

GCT

335

Leu 95

Phe

Leu

Gly

Aen

Ale 100

Gly Thr Ale

Met

Arg 105

Pro Leu

Thr

Ale

Ala HO

gtt

ACT

GCT

GCT

GCT

GGA

AAT GCA ACT

TAC

CTG

CTT GAT

GGA

CTA

CCA

383

Val

Thr

Ale

Ale

Gly 115

Gly

Asn Ale Thr

Tyr 120

Vel

leu Asp

Gly

Vel 125

Pro

AGA

ATG

AGG

GAG

AGA

ccc

ATT GGC GAC

TTG

GTT

GTC GGA

TTG

AAG

CAG

431

Arg

Met

Arg

Glu 130

Arg

Pro

lie Gly XXP 135

Leu

Vel

Vel Gly

Leu 140

Lys

Gin

err

GCT

GCA

GAT

GTT

GAT

TCT TTC CTT

GGC

ACT

GAC TGC

CCA

CCT

CTT

47»

leu

Gly

Ale US

Asp

Val

Asp

Cys Phe Leu 150

Gly

Thr

Asp Cys 155

Pro

Pro

Vel

CTT

CTC

AAT

GGA

ATC

GGA

GGG CTA CCT

GCT

GGC

AAG CTC

AAG

CTG

TCT

527

Arg

Vel 160

Aen

Gly

Xie

Gly

Gly leu Pro 165

Gly

Gly

Lys Val 170

Lys

leu

Ser

GGC

TCC

ATC

ASC

ACT

CAG

TAC TTG ACT

GCC

TTG

CTG ATG

GCT

GCT

CCT

57 5

Gly Π5

Ser

Xie

Ser

Ser

Gin 180

Tyr Leu Ser

Ale

leu 185

Leu Met

Ala

Ala

Pro 190

TTG

GCT

CTT

GSG

GAT

CTG

GAG ATT GAA

ATC

ATT

GAT AAA

TEA

ATC

TCT

623

Lau

Ale

Leu

Gly

Asp 195

Vel

Glu lie Glu

Ila 200

lie

Asp Lys

Leu

He 205

Ser

ATT

CCT

TAC

GTC

GAA

ATG

ACA TTG AGA

TTG

ATG

GAG CCT

TTT

GCT

CTG

671

lie

Pro

Tyr

Vel 210

Glu

Met

Thr Leu Arg 215

Leu

Met

Glu Arg

Phe 220

Gly

Val

ΑΛΑ

GCA

GAS

CAT

TCT

GAT

AGC TGG GAC

AGA

TTC

TAC ATT

AAG

GGA

GCT

719

Lys

Ale

Glu 225

Hie

Ser

Asp

Ser Trp Asp 230

Arg

Phe

Tyr He 235

Lys

Gly

Gly

CAA

AAA

TAC

AAG

TCC

CCT

AAA AAT GCC

TAT

CTT

GAA GCT

GAT

GCC

TCA

7 67

Gin

Lys 240

Tyr

Lye

Ser

Pro

Lys Aen Ale 245

Tyr

Vel

Glu Gly 250

Asp

Ain

Ser

AGC

GCA

ASC

TAT

TTC

TTG

GCT GCT GCT

GCA

ATT

ACT GGA

GGG

ACT

GTG

815

Ser 255

Ala

Ser

Tyr

Phe

Leu 260

Ale Gly Ale

Ala

He 265

Thr Gly

Gly

Thr

Vel 270

ACT

CTG

GAA

GCT

TCT

GGC

ACC ACC ACT

TTG

CAG

GCT GAT

CTG

AAG

y ye

863

Thr

Vel

Glu

siy

Cys 27 5

Gly

Thr Thr Ser

leu 280

Gin

Gly Asp

Val

Lys 285

Phe

GCT

GAG

GCA

CTG

GAG

ATG

ATG GGA GCG

AAG

GTT

ACA TGG

ACC

GAG

ACT

Sil

Ale

Glu

Vel

Leu 250

Glu

Met

Met Gly Ale 295

Lys

Vel

Thr Trp

Thr 300

Glu

Thr

AGC

CTA

ACT

CTT

ACT

GGC

CCA COG CGG

GAG

CCA

TTT GGG

AGG

AAA

CSC

95S

Ser

Val

Thr 305

Vel

Thr

Sly

Pro Pro Arg 310

Glu

Pro

Phe Gly 315

Arg

•Lys

Hie

CTC

AAG

GOG

ATT

GAT

GTC

AAC ATG AAC

AAG

ATG

CCT GAT

CTC

GCT

ATG

1007

Leu

Lys 320

Ale

Xie

Asp

Vel

Aen Met Asn 325

Lys

Met

Pro Asp 330

Vel

Ala

Met

ACT

CTT

GCT

GTG

GTT

GCC

CTC TTT GCC

GA7

GGC

CCT ACA

GOC

ATC

AGA

1055

Thr 335

Leu

Ale

Vel

Vel

Ale 340

Leu Phe Ala

Asp

Gly 345

Pro Thr

Ale

lie

Arg 350

GAC

GTG

GCT

TOC

TGG

AGA

CTA AAG GAG

ACC

GAG

AGG ATG

GTT

GCT

ATC

1103

Asp

Val

Alt

Ser

Trp 355

Arg

Vel Lys Glu

Thr 360

Glu

Arg Met

Vel

Ale 3«5

He

CTG

ACT

GAS

CTA

ACC

AAG

CTG GGA GCA

TCT

CTT

GAG GAA

GGG

CCT

GAC

1151

Arg

Thr

Glu

Leu

Thr

Lys

Leu Gly Ale

Mr

Val

Glu Glu

Gly

Pro

Asp

ΑΡ/Ρ/ 9 8/01 195

AP 0 u b 8 8 6

J7U

J75

JUO GCC ATC GAC

104

TAC Tyr

TOC ~Y~

ATC ATC

ac; Thr

ccc ccr; gag

AAG CTG AAC GTG

ACC Thr JV.

fie JH‘i

lie

Pru

Prj Glu 300

Lys

Leu Aen

Ya L

Ala

tie Aep

AC”

TAC

GAC

i*AC

CAC

AGG

ATG GCC

ATG

CCC TTC

T.—

•CTT

GCC

GCC TGT

1217

•ri-.r

rye

Ad'p

Aap

HU

Arj

Met Ala

Het

Ale Phe

Ser

Leu

All

Ala Cye

400

405

410

gcc

GAG

GTC

CCC

GTC

ACC

atc ax

GAG

CCT GGG

TGC

ACC

ax

AAG ACC

1295

Ad*

Glu

v»l

Pro

V«1

Thr

lie Arg

A*p

• Pro Gly

cy«

Thr

Arg

Ly» Thr

415

420

425

430

TTC

ccc

GAC

TAC

TTC

GAT

GTG CTG

AGC

ACT TTC

GTC

AAG

AAT

1337

Ph·

Pro

A*p

Tyr

Ph·

Ajrp

Yll Leu

Ser

Thr Phe

Vil

Ly»

Aen

<35

440

TAA

1340

121

INFORMATION

tor

S£Q

10 NO:3:

11) SEQUENCE

CHARACTERISTICS:

(A)

IXNGTH:

: 444 μμΙλο acid*

(BI

TYPE: anino icid

(DI

TOPOLOGY: linear

(111 MOLECULE

TYPE: protein

1x1) SEQUENCE

DESCRIPTION,

: SEQ XD »0:3:

Ala

Gly

Ala

Glu

Glu

Xie

Val Leu

Gin

Pro lie

Ly»

Glu

Xie

Ser Gly

1

5

10

15

Thr

Val

Ly»

Leu

Pro

Gly

Ser Ly»

Ser

Leu Ser

Aen

Arg

lie

Leu Leu

20

25

30

Leu

All

All

Leu

S«r

Glu

Gly Thr

Thr

Yll Vil

Aep

Am

Leu

Leu Aen

35

40

45

Ser

Glu

A»p

Val

His

Tyr

Met Leu

Gly

All Leu

Arg

Thr

Leu

Gly Leu

50

55

£0

Ser

Vil

Glu

Ail

Ly«

All All

Ly»

Arg All

Vil

Vil

Yll

Gly Cye

€5

to

75

80

Gly

Gly

Ly»

Phe

Pro

Vil

Glu Alp

All

Lye Glu

Glu

Vil

Gin

Leu Phe

85

SO

*

95

Leu

Gly

Am

All

Gly

Thr

All Met

Arg

Pro Leu

Thr

All

Ala

Vil Thr

100

105

no

All

All

Gly

ciy

Am

All

Thr Tyr

Vil

Leu Aep

Gly

Yll

Pro

Arg M»t

115

120

125

Arg

Glu

Arg

Pro

Xie

Gly

Aep Leu

Yll

Vil Gly

Leu

Ly»

Gin

Leu Gly

130

135

140

All

Axp

Vil

Aep

cy»

Phe

Leu Gly

Thr

Aep Cyw

Pre

Pro

Vil

Arg Vil

145

150

155

ICO

Am

Gly

11»

ciy

Gly

Leu

Pro Gly Gly

Lye Vil

Ly»

Leu

Ser

Gly Ser

165

170

175

lie

Ser

Ser

Gin

Tyr

Leu

S«r All

Leu

Leu Met

Ala

All

Pro

Leu Ala

l«0

its

no

Leu

Gly

Aep

Vil

Glu

Xie

Glu Xie

11»

Aep Ly»

Leu

Xie

Ser

Xie Pro

Hi

200

205

Tyr

Vil

Glu

Met

Thr

Leu

Arg Leu

Met

Glu Arg

Phe

ciy

Yll

Ly» Ala

210

215

220

Glu

HI»

Ser

Aep

Ser

Trp

Aip Arg

Phe

Tyr lie

Ly»

Gly Gly

Gin Ly»

225

230

235

240

Tyr

Ly»

Ser

Pro

Ly»

Am

All Tyr

Vil

Glu Gly

Aep

Ala

Ser

Ser Ala

245

250

255

119 5 <O oo o

δζ

6Z

ΑΡ υ ο υ 8 8 6

-.- Γ

Tyr

Ph,·

1x^4 I HO

Al.<

Gly

Al.i

Ala

tie 2*i 5

Thr

Gly

Gly

Tnr

V«L 270

Thr

Va I

a·.

Gly

Cy: I

'•I 7

Thr

Thr

Sur

Luu 2 HO

G In

Gly

Aep

Val

Ly- 2-JS

Phe

AJa

Glu

.:

* i

u i j

Me'

Met

Gly

Ala I^S

Lys

Val

Thr

Trp

Thr JOU

G Lu

The

3c r

Va .

7hr JOS

ν ί I

The

.7 I y

Pro

Pro 310

Arg

Glu

Pro

Phe

Gly 315

Arg

Lys

Hi*

Leu

Lye 320

Ala

He

Aep

Vli

Aen 325

Met

Aen

Lye

Met

Pro 330

Aep

Val

Ala

Met

Thr 335

Leu

All

Val

Val

All 34 0

Leu

Phe

Ala

Aep

Gly 34 5

Pro

Thr

Ala

Ila

Arg Aep 350

Val

Ala

Ser

Trp 355

Arg

Vil

Lye

Glu

Thr 360

Glu

Arg

Met

Vil

All 3«5

lie

Arg

Thr

Glu

Leu 370

Thr

Lye

Leu

Gly

Ala 375

Ser

Vil

Glu

Glu

Gly 380

Pro

Aep

Tyr

Cye

He 38S

He

Thr

Pro

Pro

Glu 390

Lye

Leu

Aen

Val

Thr 395

Ala

lie

Aep

Thr

Tyr 400

Aep

Aep

Hi*

Arg

Het 4 05

All

Met

Ala

Phe

Ser 410

Leu

All

Ala

eye

Ala 415

Glu

Val

Pro

v»l

Thr 420

He

Axg

Aep

Pro

Gly 425

Cye

Thr

Arg

Lye

Thr 430

Phe

Pro

Aep

Tyr

Phe

Aep

Vil

Leu

Ser

Thr

Phe

Val

Lye

Aen

(35 440 [2) INFORMATION FOR SEQ ID NO:4:

I i I SEQUENCE CHARACTERISTICS :

(Al LENGTH: 13-40 baae pair»

IB} TYPE: nucleic acid (C) STRANDEDNESS: double (DI TOPOLOGY: linear (ill MOLECULE TYPE: CDMA (vli ORIGINAL SOURCE:

(Al ORGANISM: Zea rays , (BI STRAIN: Black Hexican Sweet

-, (vill IMMEDIATE SOURCE:

I IB) CLONE: pRPA-KL-720

:..// (lx) FEATURE:

(Al NAME/KEY: CDS (BI LOCATION: 6..1337

AP/P/ 9 8/01 195 (xil SEQUENCE DESCRIPTION: SEQ ID NO:4:

CCATG GCC GGC GCC GAG GAG ATC GIG CTG CAG CCC ATC AAG GAG ATC <7

All Gly Ala Glu Glu lie Val Leu Gin Pro lie Lye Glu He

IS 10

TCC GGC ACC GTC

AAG CTG CCG GGG TCC AAG

TOG CTT TCC AAC CGG ATC Ser Leu Ser Aen Arg He

95

Ser Gly 15

Thr

Val

Lye Leu 20

Pro Gly

Ser

Lye

25

30

CPC

CTA

CTC

GCC

GCC

CTG

TOC

GAG

GGG

ACA

ACA

CTG

CTT

GAT

AAC

CTG

143

Leu

Leu

Leu

Ala

Ala

Leu

Ser

Glu

Gly

Thr

Thr

Val

Yal

Aep

Aen

Leu

35

40

45

CTG

AAC

ACT

GAG

GAT

GTC

CAC

TAC

ATG

CTC

GGG

GCC

TTG

AGG

ACT

CTT

111

Leu

A*n

Ser

Glu

Aep

Val

Hie

Tyr

Met

Leu

Gly

Ala

Leu

Arg

Thr

Leu

50

55

CO

GCT

CTC

TCT

GTC

GAA

GCG

GAC

AAA

GCT

GCC

AAA

AGA,

GCT

OTA

CTT

CTT

239

Gly

Leu

3er

Val

Glu

Ala

Aep

Lye

All

Ala

Lye

Arg

Ala

Val

Val

Val

«5

70

75

ΑΡυU Ο 8 8 6

(VC ·:ι·/

TGT C/u HI)

vrr Gly

GGA Gly

AAG Ly::

TTC Pha

CCA CTT

<ia; Glu

GAT Xup

GCT Ala

AAA CAC CAA

GTG va I

CAG Gin*

287

Pro •45

Val

Ly-· no

Glu

Glu

CTZ

TTC

TTG

<VJi

AAT

gct

criA

ATC

GCA

cry:

TCC

TTG

ACA

(ZIA

GCT

JJS

L»*u

Pht·

Luu

;iy

Λ. i.

Air

01 y

[ lr

All

Met

Ara

S«r

i^c-U

Th r

Al.n

Ala

ϊ-.

1(10

LUS

110

rm

ACT

GCT

GCT

GGT

GGA

AAT

GCA

ACT

TAC

CTG

CTT

GAT

GGA

GTA

CCA

38J

7« 1

Thr

Ala

Ala

Gly

Gly

Aan

Ala

Thr

Tyr

Val

Lau

Aap

Gly

Val

Pro

115

120

125

AGA

ATG

AGG

GAG

AGA

ccc

ATT

GGC

GAC

TTG

GTT

GTC

GGA

TTG

AAG

CAG

431

Arg

M«C

Arg

Glu

Arg

Pro

Ila

Gly

Aap

Leu

Val

Val

Gly

Lau

Lya

Gin

130

135

140

err

GGT

GCA

GAT

GTT

GAT

TGT

TTC

CTT

GGC

ACT

GAC

TGC

CCA

OCT

GTT

479

Lau

Gly

Ala

Aap

Val

A»P

cy»

Pha

Lau

Gly

Thr

Aap

cy»

Pro

Pro

Val

145

150

155

CCT

GTC

AAT

GGA

ATC

GGA

GGG

CTA

CCT

GGT

GGC

AAG

GTC

AAG

CTG

TCT

527

Arg

Val

Aan

Gly

Ila

Gly

Gly

lau

Pro

Gly

Gly

Lya

Val

Lya

Lau

Sar

160

165

170

GGC

TCC

ATC

AGC

AGT

CAG

TAC

TTG

AGT

GCC

TTG

CTG

ATG

GCT

GCT

CCT

575

Gly

5ar

Ila

Sar

Sar

Gin

Tyr

Lau

Sar

Ala

Lau

Lau

Mat

Ala

Ala

Pro

Π5

180

185

190

TTG

GCT

CTT

GGG

GAT

GTS

GAG

ATT

GAA

ATC

ATT

GAT

AAA

TTA

ATC

TCC

623

in

Lau

Ala

Lau

Gly

Aap

Val

Glu

Ila

Glu

Ila

Ila

Aap

Ly»

Lau

Ila

Sar

195

200

205

U?

ATT

CCG

TAC

GTC

GAA

ATG

ACA

TTG

AGA

TTG

ATG

GAG

CCT

TTT

GGT

GTG

671

11«

Pro

Tyr

Val

Glu

Mat

Thr

lau

Arg

Lau

Mac

Glu

Arg

Pha

Gly

Val

V—

210

215

220

O

AAA

GCA

GAG

CAT

TCT

GAT

AGC

TGG

GAC

AGA

TTC

TAC

ATT

AAG

GGA

GGT

719

Lys

Al*

Glu

Hia

Sar

Aap

Sar

Trp

Aap

Arg

Phe

Tyr

Ila

Lya

Gly Gly

225

230

235

CO

CAA

AAA

TAC

AAG

TCC

CCT

AAA

AAT

GCC

TAT

GTT

GAA

GGT

GAT

GCC

TCA

767

Gin

Lya

Tyr

Lya

Sar

Pro

Lya

Ain

Ala

Tyr

Val

Glu

Gly

Aap

Ala

Sar

240

245

250

•v-

AGC

GCA

AGC

TAT

TTC

TIG

GGT

GCT

GCA

ATT

ACT

GGA

GGG

ACT

GTG

815

Sar

Ala

Sar

Tyr

Phe

Lau

Ala

Gly

Ala

Ala

Ila

Thr

Gly

Gly

Thr

Val

6?

255

260

265

270

ACT

GTG

GAA

GGT

TGT

GGC

ACC

ACC

AGT

TTG

CAG

GGT

GAT

GTG

AAG

TTT

863

<4

Thr

V«1

Glu

Gly

Cya

Gly

Thr

Thr

Sar

Lau

Gin

Gly

Aap

Val

Lya

Pha

-

275

260

265

GCT

GAG

GTA

CTG

GAG

ATG

ATG

GGA

GCG

AAG

GTT

ACA

TGG

ACC

GAG

ACT

911

Al*

Glu

Val

Lau

Glu

Mat

Mac

Gly

Ala

Lya

Val

Thr

Trp

Thr

Glu

Thr

290

295

300

AGC

GTA

ACT

GTT

ACT

GGC

CCA

CCG

CGG

GAG

CCA

TTT

GGG

AGG

AAA

CAC

959

Sar

Val

Thr

Val

Thr

Gly

Pro

Pro

Arg

Glu

Pro

Pha

Gly

Arg

Lya

His

305

310

315

CTC

AAG

GCG

ATT

GAT

GTC

AAC

ATG

AAC

AAG

ATG

CCT

GAT

GTC

GCC

ATG

1007

Lau

Lya

Ala

Ila

Aap

Val

Aan

Mac

Aan

Lya

Mat

Pro

Aap

Val

Ala

Mat

320

325

330

ACT

err

GCT

GTG

GTT

GCC

CTC

TTT

GCC

GAT

GGC

CCG

ACA

GCC

ATC

AGA

1055

Thr

Lau

Ala

Val

Val

Ala

Lau

Pha

Ala

Aap

Gly

Pro

Thr

Ala

Ila

Arg

335

340

345

350

GAG

GTG

GCT

TCC

TGG

AGA

GTA

AAG

GAG

ACC

GAG

AGG

ATG

GTT

GCG

ATC

1103

Aap

Val

Ala

Sar

Trp

Arg

Val

Lya

Glu

Thr

Glu

Arg

Hat

Val

Ala

He

355

360

365

CGG

ACG

GAG

CTA

ACC

AAG

CTG

GGA

GCA

TCT

GTT

GAG

GAA

GGG

CCG

GAC

1151

Arg

Thr

Glu

Lau

Thr

Lya

Lau

Gly

Ala

Sar

Val

Glu

Glu

Gly

Pro

A*P

370

375

380

TAC

TGC

ATC

ATC

AGS

CCG

CCG

GAS

AAG

CTG

AAC

GTG

ACS

GCG

ATC

GAC

1199

Tyr

cy»

Ila

Ila

Thr

Pro

Pro

Glu

Lya

Lau

Aan

Val

Thr

Ala

Ila

A*P

385

390

395

AP U Ο 0 8 8 6

act; Thr	TAC Tyr ion	GAC <yc CAC	ADO Arg	AW Hwt 405	GCG Ala	AW N«t	GCC Ala	TTC TCC CTT CCC CCC TGT
A.p		Phe Ser 410	Leu Ala	Ala Cya
OCT	OAT,	GTC	CCC 'TTC	ACC	ATC	COG	GAC	CCT	G<W TOC	ACC COO	AAG ACC
Αι ι	Glu	Val	Pr-J v.it	Thr	lie	Arg	A^p	Pro	Gly Cya	Thr Arg	Lys Thr
4’.5				•120					4 25		4 30
TTC	CCC	GAC	TAC TTC	GAT	GTG	CTG	AGC	ACT	TTC GTC	AAG AAT
Phe	Pro	Aep	Tyr Pti*	Aap	Val	Leu	s«r	Thr	Phe Val	Lya Aan
			4 JS					440
ΤΑΑ
121	INFORMATION FOR	SEQ	ID NO:5:
	III SEQUENCE	CHARACTERISTICS:
			IAI LENGTH:	: 444 anino
			(Bl TYPE: anlno acid
			10) TOPOLOGY: linear
	111) MOLECULE	TYPE: protein
	(xi) SEQUENCE	DESCRIPTION:	: SEQ ID	MO:5:
Ala	Gly	Ala	Glu Glu	lie	Val	Leu	Gin	Pro	11« Ly»	Glu Ila	S»r Gly
1			5					10			IS
Thr	Val	Ly»	Leu Pro	Gly	Ser	Ly»	Ser	Leu	Ser Asn	Arg 11»	Leu Leu
			20				25			30
Lau	Ala	Ala	Leu Ser	GLu	Gly	Thr	Thr	Val	Val Aap	Am Leu	Leu Aan
		35				40				45
S«r	Glu	Aap	Val His	Tyr	Het	Leu	Gly	Ala	Leu Arg	Thr Lau	Sly Lau
	50				SS				60
s«r	Val	Glu	Ala Ajrp	Ly»	Ala	Ala	Ly»	Arg	Ala Val	Val Val	Gly Cya
65				70					75		30
Gly	Gly	Lya	Phe Pro	Val	Glu	Aap	Ala	Ly»	Glu Glu	Val Gin	Leu Phe
			35					90			95
Leu	Gly	Aan	Ala Gly	lie	Ala	Met	Arg	Ser	leu Thr	Ala Ala	Val Thr
			100				105			110
Ala	Ala	Gly	Gly Aan	Ala	Thr	Tyr	Val	Leu	Aap Gly	Val Pro	Arg Met
		115				120				125
Arg	Glu	Arg	Pro Ila	Gly	Aap	Leu	Val	Val	Gly leu	Lys Gin	leu Gly
	130				135				140
Ala	Aap	Val	Aap Cya	Phe	Leu	Gly	Thr	Aap	Cya Pro	Pro Val	Arg Val
145				150					155		160
Am	Gly	lie	Gly Gly	Leu	Pro	Gly Gly	Ly»	Val Lys	leu Ser	Gly Ser
			165					170			175
He	Ser	Ser	Gin Tyr	Leu	Ser	Ala	Leu	Leu	Met Ala	Ala Pro	Leu Ala
			ISO				185			190
Leu	Gly	Aep	Val Glu	lie	Glu	lie	lie	Aap	Lya Leu	He Ser	lie Pro
		1S5				200				205
Tyr	Val	Glu	Het Thr	Leu	Arg	Leu	Ket	Glu	Arg Phe	Gly Val	Ly» Ala
	210				215				220
Glu	Hie	Ser	Aap Ser	Trp	Aap	Arg	Phe	Tyr	Π» Lyw Gly Gly	Gin Lye
225				230					235		240
Tyr	Lye	Ser	Pro Lya	Aan	Ala	Tyr	Val	Glu	aly Aap	Ala Ser	Ser Ala
			245					250			255
Ser	Tyr	Phe	Leu Ala	Gly	Ala	Ala	lie	Thr	Gly Gly	Thr Val	Thr Val
			260				265			270
Glu	Gly	Cy»	Gly Thr	Thr	Ser	Leu	Gin	Gly	Aap Val	Ly» Phe	Ala Glu
		275				280				285

1247

12®5

1JJ7

134 0

ΑΡ/’Ρ/ 98/0119 5

APO00886

Va I

Luu ZOO

Glu

Met

Her

Gly

Ala 295

Lyw

Val

Thr

Trp

Thr 300

Glu

Thr

S«r

Va I

Thr JO*.

7-11

Thr

Gly

Pro

Pro J10

An

Glu

Pro

Pho

Gly J15

Arg

Lyo

His

Leu

Ly; J20

All

. ir

A-P

71 .

Ann J25

Hot

Asn

Lys

Hot

Pro JJ0

Asp

Val

Ala

Het

Thr JJ5

Leu

Al·

Val

V· I

Ala J<0

L«u

Ph·

Ala

Asp

Gly 3<5

Pro

Thr

Ala

II·

Arg 350

Asp

Val

Alt

s« r

Trp 355

Arg

Val

Lys

Glu

Thr 380

Glu

Arg

Het

Val

Ala 385

XI·

Arg

Thr

Glu

Lau 370

Thr

Lys

Leu

Gly

Ala 375

S«r

Val

Glu

Glu

Gly 380

Pro

Asp

Tyr

Cyw

II· 385

II·

Thr

Pro

Pro

Glu 390

Ly»

Lau

Asn

V«1

Thr 395

Ala

II·

Asp

Thr

Tyr 400

Asp

Asp

His

Arg

Net <05

Ala

Het

Ala

Ph·

S«r <10

Lau

Ala

Ala

cys

Ala <15

Glu

Val

Pro

V«1

Thr <20

II·

Arg

Asp

Pro

Gly Cys *25

Thr

Arg

Lys

Thr <30

Ph·

Pro

Asp

Tyr

Ph· <35

Asp

Val

Lau

Sar

Thr <<0

Ph·

Val

Lys

Asn

AP/P/ 9 8/01 195

Claims (15)

1. A DNA sequence coding for a mutated 5-enolpyruvylshikimate-35 phosphate synthase (EPSPS), characterized in that the EPSPS is from maize and comprises a first mutation consisting in the threonine 102 -> isoleucine substitution and a second mutation consisting in a substitution of proline 106 by serine.

2. DNA sequence coding for a mutated 5-enolpyruvylshikimate-310 phosphate synthase (EPSPS), characterised in that it comprises the coding region of the DNA sequence represented in SEQ ID No. 2, comprising a first mutation consisting in the threonine 102 isoleucine substitution and a second mutation consisting in a substitution of proline 106 by serine.

15

3. DNA sequence coding for a mutated 5-enolpyruvylshikimate -3phosphate synthase (EPSPS), characterised in that it comprises the coding sequence of SEQ ID No. 4.

4. A mutated 5-enolpyruvylshikimate -3-phosphate synthase (EPSPS),

20 characterized in that the EPSPS is maize and comprises a first mutation consisting in

-¾ the substitution of threonine 102 by isoleucine and a second mutation consisting in a

V.' substitution of proline 106 by serine.

5. A mutated 5-enolpyruvylshikimate -3-phosphate synthase (EPSPS),

25 characterized in that it comprises the peptide sequence of SEQ ID No. 5.

6. Chimeric gene comprising a coding sequence as well as regulatory elements at position 5' and 3' which are heterologous and capable of functioning in plants, characterized in that it comprises as coding sequence at least one sequence

30 according to any one of claims 1 to 3.

ΑΡ/ΓΖ 98/01195

ΑΡ ο Ο ο 8 8 6

-387. Chimeric gene according to claim 6, characterized in that it comprises a plant virus promoter.

8. Chimeric gene according to claim 6, characterized in that it 5 comprises a plant promoter.

9. Chimeric gene according to claim 8 in which the plant promoter is α-tubulin, histone, intron or actin.

10 10. Chimeric gene according to any one of claims 6 to 9, characterised i- .¾ ’to in that it comprises a zone coding for a transit peptide.

11. Chimeric gene according to claim 10, characterized in that it comprises one or more transit peptide units.

12. Vector for the transformation of plants, characterized in that it comprises at least one gene according to any one of claims 6 to 11.

13. Plant cell, characterized in that it comprises at least one gene 20 according to any one of claims 6 to 11.

25

14 . Method for the production of plants with improved tolerance to a herbicide having EPSP synthase as its target, characterised in that plant cells or protoplasts are transformed with a gene according to any one of claims 6 to 11, and in that the transformed cells are subjected to a regeneration.

30

15 Method as claimed in claim 14, characterised in that the plants with improved tolerance.are used for the preparation of hybrid lines.

ΑΡ ϋΟ ϋ 8 8 6

16. Method of treatment of plants with a herbicide having EPSPS as its target, characterized in that the herbicide is applied to plants comprising plant cells according to Claim 13.

1 ₇. Method according to claim g ₆, characterized in that glyphosate or a glyphosate precursor is applied.