The present invention relates to a new 5-enolpyruvylshikimate-3-phosphate synthase (or EPSPS) which displays increased tolerance with respect to herbicides which are competitive inhibitors with respect to phosphoenolpyruvate (PEP) of EPSPS activity. This more tolerant EPSP synthase possesses at least one xe2x80x9cthreonine by isoleucinexe2x80x9d 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 and fosametine are broad-spectrum systemic herbicides of the phosphonomethylglycine family. They act essentially as competitive inhibitors of 5-enolpyruvylshikimate-3-phosphate synthase (EC 2.5.1.19) or EPSPS with respect to the PEP (phosphoenolpyruvate). After their application to the plant, they are translocated in the plant where they accumulate in the rapidly growing parts, in particular the cauline and root spices, causing damage to the point of destruction of sensitive plants.
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 plant or bacterial origin, which is mutated or 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.
It is known, for example from U.S. Pat. No. 4,535,060, to confer on a plant a tolerance to a herbicide of the above type, especially N-phosphonomethylglycine or glyphosate, by introducing into the 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.
In the present description, xe2x80x9cplantxe2x80x9d is understood to mean any differentiated multicellular organism capable of photosynthesis, and xe2x80x9cplant cellxe2x80x9d is understood to mean any cell originating from a plant and capable of constituting undifferentiated tissues such as calluses or differentiated tissues such as embryos or plant parts or seeds.
The subject of the present invention is the production of transformed plants having increased tolerance to herbicides of the phosphonomethylglycine family, by regeneration of cells transformed by means of new chimeric genes containing a gene for tolerance to these herbicides.
The subject of the invention is also a chimeric gene for conferring on plants increased tolerance with respect to a herbicide having EPSPS as its target, comprising, 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 3xe2x80x2 end, characterized in that the glyphosate tolerance gene contains, relative to the gene from which it is derived, a xe2x80x9cthreonine 102 by isoleucinexe2x80x9d substitution in the xe2x80x9caroAxe2x80x9d (EPSPS) region. Preferably, it comprises, in addition, in the same region, a xe2x80x9cproline 106 by serinexe2x80x9d substitution. These substitutions can be introduced or be present in an EPSPS sequence of any 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 AP 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 be advantageously composed of at least one gene promoter or promoter fragment which is expressed naturally in plants (tubulin, introns, actin, histone).
The untranslated transcription termination signal region at the 3xe2x80x2 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 above, 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.
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 xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d 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 xe2x80x9cref. CPMBxe2x80x9d). 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 xcex DNA, either as a 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.