MXPA97009310A - Dna sequence of a hydroxypenyl-piruvate-dioxygenase gene and obtaining plants containing a gene of hydroxypenyl-piruvate-dioxygenase, tolerants at certain herbici - Google Patents

Abstract

A DNA sequence of a hydroxyphenyl-pyruvate-dioxygenase gene and the production of plants containing a hydroxyphenyl-pyruvate dioxygenase gene resistant to herbicides are described. The DNA sequence of a hydroxyphenyl-pyruvate-dioxygenase gene, the isolation from a bacterium or from a plant, the use to obtain herbicide-tolerant plants

Description

DNA SEQUENCE OF A GENE OF HYDROXYPENYL-PIRUVATE-DIOXYGENASE AND OBTAINING PLANTS CONTAINING A GENE OF HYDROXYPENYL -PIRUVATE-DIOXYGENASE, TOLERANTS TO CERTAIN HERBICIDES.

DESCRIPTION OF THE INVENTION The present invention relates to a hydroxyphenyl-pyruvate-dioxygenase (HPPD) gene, a chimeric gene that contains this gene as a "coding sequence, and its use for obtaining plants resistant to certain herbicides. the isoxazoles described mainly in the French patent applications Nos. 95 06800 and 95 13570 and mainly the isoxaflutole, maize selective herbicide, the dicetonitriles such as those described in the European applications 0 496 630, 0496 631, in particular the 2-cyano -3-cyclopropyl-1- (2-S02CH3-4-CF3-phenyl) -propan-1,3-dione and 2-cyano-3-cyclopropyl-1- (2- SO-CH3-2, 3-Cl2 phenyl) -propan-1,3-dione, the tricetones described in European applications 0 625 505 and 0 625 508, in particular sulcotrione. However, no tolerance gene for such herbicides has been described yet. REF: 26299 Hydroxyphenyl-pyruvate-dioxygenase is an enzyme that catalyzes the transformation reaction of parahydroxyphenyl-pyruvate into homogentisate. On the other hand, the amino acid sequence of the hydroxyphenyl-pyruvate-dioxygenase from Pseudomonas sp. P.J. 874 has been described, without there being any description of its role in the tolerance of plants to herbicides (Rüestschi et al .: Eur. J. Biochem 205, 459-466, 1992). This document does not give a description of the gene that codes for this protein. The sequence of such a gene has now been discovered and such a sequence could, once incorporated into plant cells, provide overexpression or activation of HPPD in plants, conferring to the latter an interesting tolerance against certain herbicides. recent, such as those of the ioxoxoles family or those of the tricetones. The present invention aims at a DNA sequence of a gene of non-human origin and of a non-marine bacterial origin, or even of a plant gene, isolated, or a sequence that can hybridize with that isolated sequence, characterized in that it expresses a hydroxyphenyl-pyruvate-dioxygenase (HPPD).

More particularly, the sequence can be of bacterial origin, mainly such as the genus Pseudomonas s or even of vegetable origin, mainly of monocotyledonous or dicotyledonous plants, mainly of Arabi dopsi or umbelliferae such as, for example, the carrot (Da ucus carot ta). This can be native or wild or possibly mutated, always preserving fundamentally a herbicidal tolerance property against HPPD inhibitors, such as the herbicides of the family of isoxasols or those of the tricetones. The invention also comprises a method for isolating the aforementioned gene, characterized in that: - any oligonucleotides from the amino acid sequence of a HPPD are chosen, as primers, - from these primers, the amplification fragments are synthesized by PCR - is isolated in gene by creation and selection of a genomic bank and - the gene is cloned. Preferably, primers from the HPPD sequence of a bacterium of the genus Pseudomonas are used. Particularly preferred, these are from Pseudomonas fl uorescens. The invention also has as its objective the use of a gene coding for HPPD in a process for the transformation of plants, such as the marker gene or as a coding sequence that allows conferring to the plant a tolerance to certain herbicides. It can also be used in association with other marker genes and / or coding sequence for an agronomic property. The coding gene can be of any origin, native or wild, or possibly imitated always preserved fundamentally a property of herbicidal tolerance against inhibitors of HPPD, such as the herbicides of the family of isoxasols or those of the tricetones. As the coding sequence, it is possible to use mainly those according to the invention as described above. The transformation of plant cells can be obtained by any known, appropriate means. A series of methods consists in bombarding the cells or protoplasts with particles to which the DNA sequences are anchored or joined.

Another series of methods consists in using as a means of transfer in the plant, a chimeric gene inserted in a Ti plasmid of Agrobac t eri um t umefaci ens or Ri of Agroba c t eri um rhi zogenes. The subject of the present invention is also a chimeric gene comprising, in the sense of transcription, at least one promoter regulatory sequence, a heterologous coding sequence expressing hydroxyphenyl-pyruvate-dioxygenase and at least one ternary regulatory sequence or of polyadenylation. As the promoter regulatory sequence, any promoter sequence of a gene that is naturally expressed in plants, in particular a promoter of bacterial, viral or plant origin such as, for example, that of a gene of the small subunit of ribulose can be used. -biscarboxylase (RuBisCo) or that of a gene of a-tubulin (European application EP No. 0 652 286), or even a plant virus gene such as, for example, that of the cauliflower mosaic (CAMV 195 or 35S), but any suitable, known promoter can be used. Preferably, a promoter regulatory sequence has been used that favors the overexpression of the coding sequence, such as, for example, that which comprises at least one histone promoter as described in the European application EP 0507698. According to the invention, it is also possible to use, in association with the promoter regulatory sequence, other regulatory sequences, which are located between the promoter and the promoter. the coding sequence, such as the "enhancer" transcriptional activators, such as, for example, the translation activator of the tobacco rust virus (TEV) described in the application WO87 / 07644, or transit peptides, whether simple , or double, and in this case, eventually separated by an intermediate sequence, that is to say comprising, in the sense of transcription, a sequence encoding a transit peptide of a plant gene encoding an enzyme for plastid localization , a sequence part of the mature N-terminal part of a plant gene coding for a plastidial localization enzyme, after a sequence encoding for a second transit peptide of a plant gene encoding a plastidial localization enzyme, consisting of a sequence part of the mature N-terminal part of a plant gene encoding a plastid localization enzyme, as described in European application No. 0 508 909.

As a terminator or polyadenylation regulation sequence, any corresponding sequence of bacterial origin can be used, such as for example the terminator of Agrobact eri um t umefaci ens, or even of plant origin, such as, for example, a histone terminator as described in the European application EP No. 0 633 317. The present invention even has as an objective the plant cells of monocotyledonous or dicotyledonous plants, mainly of crops, transformed according to one of the procedures described above, and which contain in their genome a effective amount of a gene expressing hydroxyphenyl-pyruvate-dioxygenase (HPPD). It has been observed that the plants transformed in this manner have an important tolerance to certain recent herbicides, such as the isoxasols described mainly in the French patent applications Nos. 95 06800 and 95 13570 and mainly the 4- [4-CF3-2- (methylsulfonyl) benzoyl] -5-cyclopropyl-isoxasol, and especially isoxaflurotol, corn selective herbicide, dicetonitriles such as those described in European applications 0 496 639, 0 496 631, in particular 2-cyano-3-cyclopropyl- l- (2-S02CH3-4-CF3phenyl) -propan-1,3-dione and 2-cyano-3-cyclopropyl-1- (2-SO-CH3- 4-2,3-Cl-phenyl) -propan-1,3-dione, the tricetones described in European applications 0 625 505 and 0 625 508, in particular sulcotrione. Finally, the invention aims at a method of weeding plants, mainly of crops, with the help of a herbicide of this type, characterized in that this herbicide is applied on plants transformed according to the invention, both in pre-seeding, in sample pre-sampling and in post-sampling of the crop. The invention even aims to use the HPPD gene as a marker gene in the course of the "transformation-regeneration" cycle of a plant species and selection on the aforementioned herbicide. The different aspects of the invention will be better understood with the help of the following experimental examples.

Example 1: Isolation of the HPPD gene from P. fl uorescens A32.

From the amino acid sequence of the HPPD of Pseudomonas sp. P.J. 874 (published by Rüetschi u et al 1992, Eur. J. Biochem. 205: 459-466), the sequence of different oligonucleotides is deduced to amplify by PCR a part of the HPPD coding sequence of P. fl uorescens A32 (isolated by McKellar, R.C. 1982, J. Appl Bacteriol, 53: 305-316). An amplification fragment of this HPPD gene has been used to select a partial genomic bank of P. fl uorescens A32, and thus isolate the gene that codes for this enzyme.

A) Preparation of the genomic DNA of P fl uorescens A32.

The bacterium has been cultivated in 40 ml of M63 minimum medium (KH2P04 13.6 g / 1, (NH4) 2S04 0.2 g / 1, MgSO4 0.2 g / 1, FeSC 0.005 g / 1 pH 7 plus L-tyrosine 10 mM as the only source carbon) at 28 ° C for 48 hours. After washing, the cells are collected in 1 ml of lysis buffer (100 mM tris-HCl pH 8.3, 1.4 M NaCl and 10 mM EDTA) and incubated 10 minutes at 65 ° C. After a treatment with phenol / chloroform (24/1) and a treatment with chloroform, the nucleic acids are precipitated by the addition of a volume of isopropanol, and then they are collected in 300 μl of sterile water and treated with RNase 10. μm / ml final. The DNA is again treated with phenol / chloroform, with chloroform and re-precipitated by the addition of 1/10 by volume of 3M sodium acetate pH 5, and 2 volumes of ethanol. The DNA is then collected in sterile, dosed water.

B) Choice of oligonucleotides and synthesis.

From the amino acid sequence of the HPPD of Pseudomonas sp P.J. 874, five oligonucleotides are chosen, two directed in the NH2 terminal sense of the protein towards the COOH end of the protenine, and three directed in the reverse direction (see figure 1). The choice has been dictated by the following two rules: - a 3 'end of the stable oligonucleotide, ie at least two bases without ambiguity. - the weakest possible degeneration. The oligonucleotides chosen have the following sequences: Pl 5'TA (C / T) GA (G / A) AA (C / T) CCIATGGG3 'P2 5'GA (G / A) ACIGGICCIATGGA3' P3 5'AA (C / T) TGCATIA (G / A) ( G / A) AA (C / T) TC (C / T) TC3 'P4 5'AAIGCIAC (G / A) TG (C / T) TG (T / G / A) ATICC3' P5 5'GC (C / T) T) TT (A / G) AA (A / G) TTICC (C / T) TCICC3 ' These have been synthesized on the MILLPORE brand "Clyclone plus DNA Synthesizer" synthesizer. With these five oligonucleotides by PCR, the amplification fragments that should theoretically be obtained after SEQ ID No. 1, have the following sizes: with the primers Pl and P3 approximately 690 bp with the primers Pl and P4 »• approximately 720 bp with the primers Pl and P5 > approximately 1000 bp with primers P2 and P3 approximately 390 bp with primers P2 and P4 * approximately 420 bp with primers P2 and P5 > approximately 700 bp C) Amplification of a coding part of the HPPD of P. fl uorescens A32.

The amplifications were performed on a PERKIN ELMER 9600 PCR apparatus and with the Taq polymerase from PERKIN ELMER, they are its buffer under standard conditions, that is, for 50 μl of reaction there are the dNTPs at 200 μM, the primers at 20 μM, 2.5 units of Taq polymerase and 2.5 μg of P DNA. fl uorescens A32.

The amplification program used is, 5 minutes at 95 ° C plus 35 cycles at < 45 sec at 95 ° C, 45 sec. at 49 ° C, 1 min. at 72 ° C > followed by 5 minutes at 72 ° C. Under these conditions, all the amplification fragments obtained have a size compatible with the theoretical sizes given above, which is a good indication of the specificity of the amplifications. The amplification fragments obtained with the primer sets P1 / P4, P1 / P5 and P2 / P4 are ligated into pBSII SK (-) after digestion of this plasmid with EcoRV and treatment with the terminal transferase in the presence of ddTTP as described in HOLTON TA and GRAHAM MW 1991. NAR vol. 19, No. p 1156. A clone of each of these three types is partially sequenced; This confirms that it has been well amplified in all three cases in a part of the coding region of the HPPD of P. fl uorescens A32. The P1 / P4 fragment is retained as a probe to select a partial genomic bank of P. fl uorescens A32, and isolate the entire HPPD gene.

D) Isolation of the gene.

By Southern blotting, it is shown that a 7 Kpb fragment after digestion of P. fl uorescens A32 DNA with the BamHI restriction sign, hybridizes with the HPPD P1 / P4 probe. 400 μg of DNA of P. fl uorescens A32 were then digested with the restriction enzyme BamHI and DNA fragments which are approximately 7 Kpb are purified on an agarose gel. These fragments are ligated into pBSII SK (-), likewise digested with BamHI and dephosphorylated by treatment with alkaline phosphatase. After transformation into E. coli DHlOb, the partial genomic bank is selected with the HPPD P1 / P4 probe. A positive clone has been isolated and called pRP A. Its simplified letter is given in figure 2. On this chart or map the position of the coding part of the HPPD gene is indicated. This is composed of 1077 nucleotides coding for 358 amino acids (see SEQ ID No. 1). The HPPD of P. fl uorescens A32 shows a good homology of amino acids with that of Pseudomonas sp strain P.J.874, there being in fact 92% identity between these two proteins (see figure 3).

Example 2: Construction of two chimeric genes To confer the tolerance of the plants to the HPPD-inhibiting herbicides, two chimeric genes are constructed: The first is to put the coding part of the HPPD gene of P. fl uorescens A32 under the control of the double histone promoter (Request European Patent No. 0 507 698) followed by the traditional tobacco mosaic virus (TEV) enhancer (pRTL-GUS (Carrington and Freed, 1990; J. Virol. 64: 1590-1597)) with the terminator of the nopalin-synthase. The HPPD will then be localized in the cytoplasm. The second one will be identical to the first one, closer than between the activator of the TEV translation and the coding part of the HPPD, the optimized transit peptide (OTP) is inserted (European Application EP No. 0 508 909). The HPPD will then be located in the chloroplast.

A) Construction of the vector pRPA-RD-153 - pRPA-RD-11: A derivative of pBS-II SK (-) (Stratagene catalog # 212206) containing the polyadenylation site of nopalin-synthase (NOS polyA) (European Application EP No. 0 652 286) is cloned between the Kpnl and Salí sites. The Kpnl site is transformed into a Notl site by treatment with T4 DNA polymerase I in the presence of 150 μm of deoxucleotide triphosphates and then ligation with a Notl linker (Stratagene catalog # 1029). This gives a NOS polyA cloning cassette. pRPA-RD-127: A derivative of pRPA-BL-466 (European application EP No. 0 337 899) cloned in pRPA-RD-11 creating an expression cassette of the oxy gene and containing the promoter of the small subunit of ribulose-biscarboxylase: "promoter (SSU) -gen-oxy- NOS poliA "To create this plasmid, pRPA-BL-488 has been digested with Xbal and HindIII to isolate a fragment of 1. 9 kbp containing the SSU promoter and the oxy gene that has been ligated into the plasmid pRPA-RD-11 digested with the compatible enzymes. - pRPA-RD-132: is a derivative of pRPA-BL-488 (European Application EP No. 0 507 698) cloned in pRPA-RD-127 with the creation of an expression cassette of the oxy gene with the double histone promoter : double histone promoter - oxy gene - NOS poliA " To make this plasmid, pRPA-BL-466 is digested with HindIII treated with Klenow and then digested again with Ncol. The purified 1.35 kbp fragment containing the double promoter of histone H3A748 is ligated with the plasmid pRPA-RD-127 which had been digested with Xbal, treated with Klenow and redigerled with Ncol. - pRPA-RD-153: is a derivative of pRPA-RD-132 that contains the translation activator of the tobacco rust virus (VTE). pRTL-GUS (Carrington and Freed 1990; J. Virol. 64: 1590-1597) is digested with Ncol and EcorI and the 150 bp fragment is ligated into pRPA-RD-132 digested with the same enzymes. Then an expression cassette containing the promoter has been created: "double histone promoter -TEV-oxy-NOS polyA gene" B) Construction of vector pRPA-RD-185: PUC19 / GECA: A derivative of pUC19 (Gibco catalog # 15364-011) containing numerous cloning sites. pUC19 is digested with EcorI and ligated with linker oligonucleotide 1: Linker 1: AATTGGGCCA GTCAGGCCGT TTAAACCCTA GGGGGCCCG CCCGGT CAGTCCGGCA AATTTGGGAT CCCCCGGGC TTAA The selected clone contains an EcoRI site followed by the polylinker containing the following sites: EcoRI, Apa l, Avrll, P el, Sfi l, Sacl, Kpnl, Sma l, BamHI, Xba l, Sal i, Ps t I, Sphl and HindIII. pRPS-RD-185: is a derivative of pUC19 / GECA that contains a modified polylinker. pUC19 / GECA is digested with HindIII and ligated with the oligonucleotide linker 2: Linker 2: AGCTTTTAAT TAAGGCGCGC CCTCGAGCCT GGTTCAGGG AAATTA ATTCCGCGCG GGAGCTCGGA CCAAGTCCC TCGA The selected clone contains a HindIII site by means of the polylinker that now contains the following sites: EcoRI, Apa l, Avrll, Pmel, Sfi l, Sacl, Kpnl, Sma l, BamHI, Xba l, Sal i, Ps t I, Sphl, HindIII, Pací, AscI, Xhol, and EcoNI.

C) Construction of the pRP T vector: pRP 0: a derivative of pRPA-RD-153 containing an HPPD expression cassette, double histone promoter-TEV-HPPD gene-terminator Nos. To manufacture pRP 0, pRPA-RD-153 is digested with HindIII, treated with the Klenow fragment and then redigerled with Ncol to take the oxy gene and replace it with the HPPD gene from the pRP A plasmid, by digestion with BstEII, Klenow treatment and digestion with Ncol again. - pRP R: to obtain the plasmid, pRP 0 has been digested with PvuII and Sacl, the chimeric gene has been purified, then ligated into pRPA-RD-185, then digested with PvuII and Sacl. pRP T: this was obtained by ligating the chimeric gene from pRP R and then digestion with Sacl and HindIII in plasmid pRPA-BL 150 alpha2, digested with the same enzymes (European Application EP No. 0 508 909). The chimeric gene of vector pRP T therefore has the following structure: Promoter of TEV Coding region of Terminator histone double HPPD us D) Construction of the vector pRP V pRP P: this is a derivative of pRPA-RD-7 (European Application EP No. 0 652 286) containing the optimized transit peptide, followed by the HPPD gene. This was obtained by ligating the coding part of HPPD from pRP A, by digestion BstEII and Ncol, treatment with Klenow and the plasmid pRPA-RD-7, this same one digested with Sphl and Accl and treated with the DNAase of the polymerase T4 - pRP Q: a derivative of pRPA-RD-153 containing a cassette for HPPD expression, double histone promoter -TEV-OTP-HPPD gene-terminator Nos. To construct the plasmid, pRPA-RD-153 is digested with I left, treated with Klenow and then redigerled with Ncol to take the oxy gene and replace it with the HPPD gene from the pRP P plasmid by BstEII digestion, Klenow treatment and Ncol redigestion. - pRP S: to obtain it, plasmid pRP Q was digested with PvuII and Sacl to obtain the chimeric gene that has been ligated in pRPA-RD-185, and then digested with PvuII and Sacl. pRP V: this was obtained by ligating the chimeric gene from pRP S, after digestion with Sacl and HindIII in the plasmid pRPA-BL-150 alpha2 (European Application EP No. 0 508 909).

The chimeric gene of vector pRP Q thus has the following structure: Example 3: Transformation of tobacco industrial PBD6.

In order to determine the efficacy of these two chimeric genes, these have been transferred in industrial tobacco PBD6 according to the transformation and regeneration procedures already described in European application EP No. 0 508 909. 1) Transformation: The vector is introduced into the non-oncogenic strain of Agrobacterium EHA 101 (Hood et al. 1987) carrier of the cosmid pTVK 291 (Komari et al. 1986). The transformation technique is based on the procedure of Horsh R. et al. (1985) Science, 227, 1229-1231. 2) Regeneration: The regeneration of tobacco PBD6 (from SEITA France) from leaf explants, is carried out on a base medium of Murashige and Skoog (MS) comprising 30 g / 1 sucrose as well as 200 μg / ml kanamycin. The leaf explants are taken from plants in greenhouse or in vitro, and transformed according to the leaf disc technique (Science 1985, Vol.227, p.1229-1231) in three successive stages: the first one comprises the induction of shoots on MS medium added of 30 g / 1 of sucrose containing 0.05 mg / l of naphthylacetic acid (ANA) and 2 mg / l of benzylaminopurine (BAP) for 15 days. The shoots formed in the course of this stage are immediately developed by cultivation in an MS medium supplemented with 30 g / 1 of sucrose but not containing hormone, for 10 days. Then the developed shoots are taken and they are grown on an MS rooting medium with a medium proportion of salts, vitamins and sugars, and that does not contain hormone. After about 15 days, the rooted shoots are passed to the ground.

Example 4: Measurement of Tobacco Tolerance to 4- [4-CF3-2-Imethylsulonyl) benzoyl] -5-cyclopropyl-isoxasol: post-uptake treatment.

Upon exiting the in vitro test, the transformed tobacco seedlings were acclimated in greenhouse (60% relative humidity, temperature 20 ° C at night, 23 ° C for the day) for five weeks and then treated with 4- [4 -CF3-2- (methylsulfonyl) benzoyl] -5-cyclopropyl-isoxasol. Control tobacco that was not transformed and treated with 4- [4-CF3-2- (methylsul-fonyl) -benzoyl] -5-cyclopropyl-isoxasol at doses ranging from 50 to 400 g / ha, developed in about 72 hours chlorosis, which intensifies to evolve to very pronounced necrosis in a week (covering approximately 80% of the terminal leaves). After transformation this same tobacco plant, which overexpresses the HPPD of P. fl uorescens, is very well protected against a treatment with 4- [4-CF3-2- (methylsulfonyl) benzoyl] -5-cyclopropyl-isoxasol at dose of 400 g / ha. If the overexpressed enzyme is in the cytoplasm, that is, if the transformation has been done with the gene carried by the vector pRP T, subsequently the plant presents very light chlorosis all located on the intermediate leaves. If the overexpressed enzyme is in the chloroplast, that is, if the transformation has been done with the gene carried by the vector pRP V, then the plant is perfectly protected, and does not present any symptoms.

Example 5: Measurement of Tobacco Tolerance to 4- [4-CF3-2- (Methylsul-fonyl) -benzoyl] -5-cyclopropyl-isoxasol: Pre-treatment a) in vitro test: Tobacco grains harvested from plants derived from the cycle of "transformation-regeneration" resistant to a foliar treatment of isoxaflutole at the dose of 400 g / ha described in examples 1 to 3 are used. These grains have been planted on boxes that They contain phytoagar at 10 g / 1 and isoxaflutole at different concentrations ranging from 0 to 1 mg / l. The germination was done immediately at 25 ° C with a photoperiod of 12 hours of light / 12 of darkness.

According to this protocol, wild tobacco grains have been put to germinate, as well as the grains of two types of transgenic tobacco, ie CY tobaccos, with HPPD localization in the cytoplasm, and CO tobaccos with HPPD the chloroplast. The measurements of the resistances are made visually between 2 and 3 weeks after sowing. The results are reported in the following table. concentration tobacco tobacco CY tobacco CO of isoxaflutol wild 0 mg / l 100% of 100% of 100% of grains grains germinate without germinate without germinate without symptoms0 symptoms0 symptoms 0. 05 mg / l 20% of J -OS 75% of the 75% of the grains grains grains germinate and germinate * without germinating * without presenting symptoms0 symptoms0 symptoms0 0.1 mg / l step of 75% of the 75% of the germination grains grains germinate * without germinating * without symptoms0 symptoms0 0. 5 mg / l step of 75% of the 75% of germination grains grains germinate * without germinating * without symptoms0 symptoms0 1 mg / l step of 75% of the 75% of germination grains grains germinate * with germinate * without slight symptoms0 symptoms0 0 the symptoms that the seedlings present in the course of germination are deformations of the cotyledons, more or less important and above all a whitening of the normally photosynthetic and therefore green tissues. * 75% of the grains germinate because they have been planted with grains from the self-fertilization of mono-locus plants that come from the cycle "transformation-regeneration" that do not include the gene of tolerance rather than a chromosome. By operating in the same way with the following products, product No. 51 of the American patent 4 780 127, the same results are obtained at a concentration of 0 mg / l and 0.1 mg / l on wild tobacco and CO tobacco. b) greenhouse test: The procedure is as in Example 4, with the difference that the treatment was carried out in pretoma, 24 hours before sowing. Wild planting is normally carried out. Under these conditions it is observed that for the seeding of untreated controls, there is no germination for the entire herbicide dose at least equal to 10 g / ha. In contrast, CY tobacco does not present any symptoms, as defined in paragraph a), up to 100 g / ha. Likewise, CO tobacco does not present any symptoms, as defined in paragraph a), up to 200 g / ha. These results clearly show that the HPPD gene of P. fl uorescens confers tolerance to tobacco against treatments in pretoma to isoxaflutole. This tolerance is better if the protein is located in the chloroplast instead of in the cytoplasm.

Example 6: With the aim of studying if the gene of HPPD of P. fl uorescens can be used as a marker gene in the course of the "transformation-regeneration" cycle of a plant species, the tobacco has been transformed with the HPPD gene and the transformed plants have been obtained after the selection about isoxaflutole.

Material, methods and results The pRP V chimeric gene described above is transferred in the industrial tobacco PBD6 according to the transformation and regeneration procedures already described in the European application EP No. 0 508 909. The chimeric gene of the vector pRP V has the following structure: Trans training: The vector is introduced into the non-oncogenic strain of Agrobac t erium EHA 101 (Hood et al. 1987) carrier of the cosmid pTVK 291 (Komari et al. 1986). The transformation technique is based on the procedure of Horsh et al. (1985). 2) Regeneration: The regeneration of PBD6 tobacco (from SEITA France) from leaf explants is performed on a Murashige and Skoog (MS) base medium comprising 30 g / 1 of sucrose as well as 350 mq / 1 of cefotaxime and 1 mg / l of isoxaflutole. The foliar explants are taken from the plants in greenhouse or in vitro, and transformed according to the leaf disc technique (Science 1985, Vol.227, p.1229-1231) in three successive stages: the first one comprises the induction of shoots on an MS medium added of 30 g / 1 of sucrose containing 0.05 mg / l of naphthylacetic acid (ANA) and 2 mg / l of benzylaminopurine (BAP) for 15 days and 1 mg / l of isoxaflutole. The green shoots formed in the course of this stage are then developed by cultivation on an MS medium supplemented with 30 g / 1 sucrose and 1 mg / l isoxaflutole, but not containing hormone, for 10 days. Then the developed explants are taken and they are grown on a rooting medium MS at an average proportion of salts, vitamins and sugars and 1 mg / l of isoxaflutole, and which does not contain hormone. After about 15 days, the rooted shoots are passed to the ground.

All the seedlings obtained according to this protocol are analyzed by PCR with the specific HPPD primers of P. fl uorescens. This PCR analysis has confirmed that all the plants obtained in this way have a well integrated HPPD gene. In conclusion, this trial confirms that the HPPD gene can be used as the marker gene and that, associated with this gene, isoxaflutole can be a good selection agent.

Examples 7 and 8: Isolation of the HPPD gene from Arabi dopsi s thal i ana and the carrot HPPD gene. { Daucus carot ta) a) Construction of cDNA banks.

The mRNAs extracted from young seedlings of Arabidopsis s thaliana, and the mRNAs extracted from carrot cells in culture, served to construct two cDNA libraries in the vector Uni Zap * R XR marketed by the company Stratagen, according to the protocol postulated by this company. b) Selection of cDNA banks.

These two libraries have been selected with the aid of a probe corresponding to a partial length Arabi dopsi s thal i ana cDNA, obtained via the Arabidopsis Biological Resource Center (Ohio, E.U.A.) and classified: EST clone No. 91B13T7. This clone consists of approximately 500 base pairs where only 228 had been sequenced by the MSU-DOE Plant Research Laboratory in the framework of the random sequencing of the Arabidopsi s thal i ana cDNA. We have completely sequenced the 500 base pairs before using this clone to select Arabidopsis thaliana and carrot cDNA libraries with the help of the classical lysis plate hybridization technique (reference?). c) An Arabidopsi s thaliana ANDc (SEQ ID No. 2) corresponding to 1338 base pairs has been obtained. East CDNA possesses a start codon of translation at position 25, and a codon at the end of translation at position 1336. This cDNA is thus complete and codes for a protein of 445 amino acids. d) A carrot cDNA has been obtained. { Da ucus ca ro t ta) (SEQ ID No. 3) corresponding to 1329 base pairs. This cDNA possesses a start codon of translation at position 1, and a codon at the end of translation at position 1329. This cDNA is thus complete and codes for a protein of 442 amino acids.

The illustrated sequences are the following: SEQ ID No. 1 Sequence of the HPPD gene of Pseudomonas fluorescens A32. SEQ ID No. 2 HPDV cDNA Sequence of Arabidopsis s Thali SEQ ID No. 3 HPPD cDNA sequence of Da ucus caro t ta The following figures are given by way of illustration to illustrate the invention.

Figure 1 represents the protein sequence of the HPPD of Pseudomonas s sp. strain P.J. 874, and the theoretical nucleotide sequence of the corresponding coding part; the five oligonucleotides chosen to carry out the amplification of a part of this coding region are symbolized by the five arrows.

Figure 2 depicts the plasmid mapping with the 7 kb genomic DNA fragment, which contains the HPPD gene of P. fl uorescens A32.

Figure 3 gives the comparison of the amino acid sequences of the HPPD of P. fl uorescens A32 and of the HPPD of Pseudomonas sp strain P.J 874 (only the divergent amino acids between the two sequences are indicated) as well as the consensual sequence. - > - • > LIST OF SEQUENCES (1. GENERAL INFORMATION (i) APPLICANT: Sailland, Alain Rolland, Anne Matringe, Michel Pallet, Kenneth E. (ii) TITLE OF THE INVENTION: DNA SEQUENCE OF A GENE OF HYDROXYPENYL-PIRUVATE-DIOXYGENASE AND OBTAINING PLANTS CONTAINING A GENE OF HYDROXYPHENYL-PIRUVATE-DIOXYGENASE, TOLERANT TO CERTAIN HERBICIDES. (iii) SEQUENCE NUMBER: 3 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Francois Chretien (B) STREET: 14-20 rue Pierre BAIZET (C) CITY: Lyon Cedex 09 (E) COUNTRY: France (F) POSTAL CODE: 69263 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible Disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentin Relay # 1.0, Version # 1.25 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: FR PH95033 (B) DATE OF SUBMISSION: 02-JUN-1995 (C) CLASSIFICATION: (vii) INFORMATION FROM THE LAWYER / AGENT (A) NAME: Chretien, Francois (ix) INFORMATION FOR TELECOMMUNICATION: (A) TELEPHONE: 72-29-26-42 (B) TELEFAX: 72-29-28-43 (2) INFORMATION FOR SEQ ID No. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1077 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: DNA (genomic) (ii! HYPOTHETICAL: NO (iv) ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas fluorescens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 1 ATGGCAGATC TATACGAAAA CCCAATGGGC CTGATGGGCT TTGAATTCAT CGAATTAGCG 6 TCGCCGACGC CGGGTACCCT GGAGCCGATC TTCGAGATCA TGGGCTTCAC CAAAGTCGCG 12 ACCCACCGTT CCAAGAACGT GCACCTGTAC CGCCAGGGCG AGATCAACCT GATCCTCAAC 19 AACGAGCCCA ACAGCATCGC CTCCTACTTT GCGGCCGAAC ACGGCCCGTC GGTGTGCGGC 24 ATGGCGTTCC GCGTGAAGGA CTCGCAAAAG GCCTACAACC GCGCCCTGGA ACTCGGCGCC 30 CAGCCGATCC ATATTGACAC CGGGCCGATG GAATTGAACC TGCCGGCGAT CAAGGGCATC 36 GGCGGCGCGC CGTTGTACCT GATCGACCGT TTCGGCGAAG GCAGCTCGAT CTACGACATC 42 GACTTCGTGT ACCTCGAAGG TGTGGAGCGC AATCCGGTCG GTGCAGGTCT CAAAGTCATC 48 GACCACCTGA CCCACAACGT CTATCGCGGC CGCATGGTCT ACTGGGCCAA CTTCTACGAG 54 AAATTGTTCA ACTTCCGTGA AGCGCGTTAC TTCGATATCA AGGGCGAGTA CACCGGCCTG 60 ACTTCCAAGG CCATGAGTGC GCCGGACGGC ATGATCCGCA TCCCGCTGAA CGAAGAGTCG 66 TCCAAGGGCG CGGGGCAGAT CGAAGAGTTC CTGATGCAGT TCAACGGCGA AGGCATCCAG 72 CACGTGGCGT TCCTCACCGA CGACCTGGTC AAGACCTGGG ACGCGTTGAA GAAAATCGGC 78 ATGCGCTTCA TGACCGCGCC GCCAGACACT TATTACGAAA TGCTCGAAGG CCGCCTGCCT 84 GACCACGGCG AGCCGGTGGA TCAACTGCAG GCACGCGGTA TCCTGCTGGA CGGATCTTCC 90 GTGGAAGGCG ACAAACGCCT GCTGCTGCAG ATCTTCTCGG AAACCCTGAT GGGCCCGGTG 96 TTCTTCGAAT TCATCCAGCG CAAGGGCGAC GATGGGTTTG GCGAGGGCAA CTTCAAGGCG 102 CTGTTCGAGT CCATCGAACG TGACCAGGTG CGTCGTGGTG TATTGACCGC CGATTAA 107 (2) INFORMATION FOR SEQ ID No. 2 (i) CHARACTERISTICS OF THE SEQUENCE: 5 (A) LENGTH: 1338 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: Linear 0 (ii) TYPE OF MOLECULE: cDNA (ii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Arabidopsis thaliana (B) CEPA: Columbia (d) DEVELOPMENT STAGE: Young green plant 0 (vii) IMMEDIATE SOURCE (A) GENOTECA: Uni zap XR STRATAGENE .'xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No. 2: ATGGGCCACC AAAACGCCGC CGTTTCAGAG AATCAAAACC ATGATGACGG CGCTGCGTCG 6 TCGCCGGGAT TCAAGCTCGT CGGATTTTCC AAGTTCGTAA GAAAGAATCC AAAGTCTGAT 12 AAATTCAAGG TTAAGCGCTT CCATCACATC GAGTTCTGGT GCGGCGACGC AACCAACGTC 18 GCTCGTCGCT TCTCCTGGGG TCTGGGGATG AGATTCTCCG CCAAATCCGA TCTTTCCACC 24 GGAAACATGG TTCACGCCTC TTACCTACTC ACCTCCGGTG ACCTCCGATT CCTTTTCACT 30 GCTCCTTACT CTCCGTCTCT CTCCGCCGGA GAGATTAAAC CGACAACCAC AGCTTCTATC 36 CCAAGTTTCG ATCACGGCTC TTGTCGTTCC TTCTTCTCTT CACATGGTCT CGGTGTTAGA 42 GCCGTTGCGA TTGAAGTAGA AGACGCAGAG TCAGCTTTCT CCATCAGTGT AGCTAATGGC 48 GCTATTCCTT CGTCGCCTCC TATCGTCCTC AATGAAGCAG TTACGATCGC TGAGGTTAAA 54 CTATACGGCG ATGTTGTTCT CCGATATGTT AGTTACAAAG CAGAAGATAC CGAAAAATCC 60 GAATTCTTGC CAGGGTTCGA GCGTGTAGAG GATGCGTCGT CGTTCCCATT GGATTATGGT 66 ATCCGGCGGC TTGACCACGC CGTGGGAAAC GTTCCTATTG TTGGTCCGGC TTTAACTTAT 72 GTAGCGGGGT TCACTGGTTT TCACCAATTC GCAGAGTTCA CAGCAGACGA CGTTGGAACC 78 GCCGAGAGCG GTTTAAATTC AGCGGTCCTG GCTAGCAATG ATGAAATGGT TCTTCTACCG 84 ATTAACGAGC CAGTGCACGG AACAAAGAGG AAGAGTCAGA TTCAGACGTA TTTGGAACAT 90 AACGAAGGCG CAGGGCTACA ACATCTGGCT CTGATGAGTG AAGACATATT CAGGACCCTG 96 AGAGAGATGA GGAAGAGGAG CAGTATTGGA GGATTCGACT TCATGCCTTC TCCTCCGCCT 102 ACTTACTACC AGAATCTCAA GAAACGGGTC GGCGACGTGC TCAGCGATGA TCAGATCAAG 108 GAGTGTGAGG AATTAGGGAT TCTTGTAGAC AGAGATGATC AAGGGACGTT GCTTCAAATC 114 TTCACAAAAC CACTAGGTGA CAGGCCGACG ATATTTATAG CGATAATCCA GAGAGTAGGA 120 TGCATGATGA AAGATGAGGA AGGGAAGGCT TACCAGAGTG GAGGATGTGG TGGTTTTGGC 126 AAAGGCAATT TCTCTGAGCT CTTCCAGTCC ATTGAAGAAT ACGAAAAGAC TCTTGAAGCC 132 AAACAGTTAG TGGGATGA 133 (2) INFORMATION FOR SEQ ID No. 3: (i) CHARACTERISTICS OF THE SEQUENCE: 5 (A) LENGTH: 1329 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear 0 (ii) TYPE OF MOLECULE: cDNA (ii) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Daucus carotta (D) DEVELOPMENT STAGE: Cells in suspension (vii) IMMEDIATE SOURCE 0 (A) GENOTECA: Uni zap XR STRATAGENE (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No 3: ATGGGGAAAA AACAATCGGA AGCTGAAATT CTCTCAAGCA ATTCATCAAA CACCTCTCCT 6 GAAACATTCA AGCTGGTCGG TTTCAACAAC TTCGTCCGCG CCAACCCCAA GTCCGATCAC 12 TTCGCCGTGA AGCGGTTCCA CCACATTGAG TTCTGGTGCG GCGACGCCAC CAACACGTCG 18 CGGCGGTTCT CGTGGGGCCT CGGCATGCCT TTGGTGGCGA AATCGGATCT CTCTACTGGA 24 AACTCTGTTC ACGCTTCTTA TCTTGTTCGC TCGGCGAATC TCAGTTTCGT CTTCACCGCT 30 CCTTACTCTC CGTCCACGAC CACTTCCTCT GGTTCAGCTG CCATCCCGTC TTTTTCGGCA 36 TCGGGTTTTC ACTCTTTTGC GGCCAAACAC GGCCTTGCTG TTCGGGCTAT TGCTCTTGAA 42 GTTGCTGACG TGGCTGCTGC GTTTGAGGCC AGTGTTGCGC GTGGGGCCAG GCCGGCTTCG 48 GCTCCTGTTG AATTGGACGA CCAGGCGTGG TTGGCTGAGG TGGAGTTGTA CGGAGATGTG 54 GTCTTGAGGT TTGTTAGTTT TGGGAGGGAG GAGGGTTTGT TTTTGCCTGG ATTCGAGGCG 60 GTGGAGGGGA CGGCGTCGTT TCCGGATTTG GATTATGGAA TTAGAAGGCT TGATCATGCG 66 GTGGGGAATG TTACCGAGTT GGGGCCTGTG GTGGAGTATA TTAAAGGGTT TACGGGGTTT 72 CATGAATTTG CGGAGTTTAC AGCGGAGGAT GTGGGGACTT TGGAGAGTGG GTTGAATTCG 79 GTGGTGTTGG CGAATAATGA GGAGATGGTT CTGTTGCCCT TGAATGGGCC TGTGTATGGG 94 ACCAAGAGGA AGAGTCAGAT ACAGACTTAC TTGGAGCACA ATGAAGGGGC TGGAGTGCAG 90 CATTTGGCTT TAGTGAGTGA GGATATTTTT AGGACTTTAA GGGAGATGAG GAAGAGGAGT 96 TGCCTTGGTG GTTTTGAGTT TATGCCTTCG CCACCGCCTA CGTATTACAA GAATTTGAAG 102 AATAGGGTCG GGGATGTGTT GAGTGATGAA CAGATCAAGG AGTGTGAAGA TTTGGGGATT 108 TTGGTGGATA GGGATGATCA GGGTACATTG CTTCAAATCT TTACCAAGCC TGTAGGTGAC 114 AGGCCTACCT TATTCATAGA GATCATTCAG AGGGTAGGGT GCATGCTCAA GGACGATGCA 120 GGGCAGATGT ACCAGAAGGG CGGGTGCGGA GGATTTGGGA AGGGGAACTT CTCAGAGCTG 126 TTCAAGTCCA TCGAAGAATA TGAAAAAACA CTTGAAGCTA AACAAATCAC TGGATCTGCT 132 GCTGCATGA 132 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 (28)

1. The sequence of a gene of non-human origin or of a non-marine, isolated bacterium, or the sequence that can hybridize with this sequence, characterized in that it expresses a hydroxyphenyl-pyruvate dioxygenase (HPPD).

2. The sequence according to claim 1, characterized in that this e-s of bacterial or plant origin.

3. The sequence according to claim 2, characterized in that it is from Pseudomonas s sp.

4. The sequence according to claim 3, characterized in that it is from Pseudomonas s fl uorescens.

5. The sequence according to claim 1, characterized in that it is of vegetable origin.

H.H. The sequence according to claim 5, characterized in that it is from Arabi dopsi s.

7. The sequence according to claim 5, characterized in that it is from an Umbellifera.

8. The method of isolation of the gene according to any of claims 1 to 4, characterized in that: selected oligonucleotides from the amino acid sequence of a HPPD are chosen as primers. - from these primers, the amplification fragments are synthesized by PCR - the gene is isolated by creation and selection of a genomic bank and - the gene is cloned.

9. The chimeric gene for the genetic transformation of plants that comprise, in the sense of transcription: at least one promoter regulatory sequence from a gene that is naturally expressed in plants, - a heterologous coding sequence, - at least one polyadenylation sequence, characterized in that the coding sequence is a sequence of a gene expressing hydroxyphenyl-pyruvate dioxygenase (HPPD).

10. The chimeric gene according to claim 9, characterized in that the promoter regulatory sequence favors the overexpression of the coding sequence.

11. The chimeric gene according to claim 10, characterized in that the promoter regulatory sequence comprises at least one histone promoter.

12. The chimeric gene according to any of claims 9 to 11, characterized in that it comprises, between the promoter regulatory sequence and the coding sequence, a transit peptide.

13. The chimeric gene according to claim 9, characterized in that it comprises, between the promoter regulatory sequence and the coding sequence, an optimized transit peptide comprising, in the direction of transcription, a coding sequence for a transit peptide of a plant gene coding for a plastidial localization enzyme, a part of the sequence of the mature N-terminal portion of a plant gene encoding a plastidial localization enzyme, subsequently a sequence encoding a second transit peptide of a gene plant that codes for a plastidial localization enzyme.

14. The chimeric gene according to claims 9 to 13, characterized in that it comprises, between the promoter regulatory sequence and the coding sequence, a sequence of a transcription activator (enhancer).

15. The vector usable for the genetic transformation of plants, characterized in that it comprises a chimeric gene according to any of claims 9 to 14.

16. A plant cell, characterized in that it comprises a chimeric gene according to any of the 9 to 14 subdivisions.

17. A plant, characterized in that it is regenerated from cells according to claim 16.

18. The plant according to claim 17, characterized in that it belongs to the family of the dicotyledons.

19. The process of transforming plants to make them tolerant to HPPD inhibitors, characterized the procedure because a gene expressing an exogenous HPPD is introduced into the plant cell.

20. The method according to claim 19, characterized in that the transfer is carried out with Agrobacterium um t umefaci ens or Agrobacteri um rhi zogenes.

21. The method according to claim 19, characterized in that the transfer It is carried out by bombardment with the help of DNA-loaded particles.

22. The plant transformation process, characterized in that a gene expressing an exogenous HPPD is introduced as a selection marker into the plant cell.

23. The plant-selective herbicidal treatment process, characterized in that an inhibitor of the HPPD gene is applied to a plant according to any of claims 17 and 18.

24. The method according to claim 23, characterized in that the inhibitor of the HPPD gene is an isoxazole.

25. The process according to claim 24, characterized in that isoxasol is 4- [4-CF3-2- (methylsulfonyl) benzoyl) -5-cyclopropyl-isoxazole.

26. The method according to claim 23, characterized in that the inhibitor of the HPPD gene is a dicetonitrile.

27. The method according to claim 23, characterized in that the inhibitor of the HPPD gene is a tricetone.

28. The method according to claim 23, characterized in that the inhibitor of the HPPD gene is a sulcotrione.