Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
1998-02-18
2001-07-31
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C800S298000, C800S278000, C800S275000, C536S023500, C536S023600, C536S023700, C536S024100, C435S468000, C435S419000
Reexamination Certificate
active
06268549
ABSTRACT:
The present invention relates to a hydroxyphenylpyruvate dioxygenase (HPPD) gene, a chimeric gene comprising this gene as coding sequence and its use to obtain plants resistant to certain herbicides.
Certain herbicides have been disclosed, such as the isoxazoles described especially in the French Patent Applications 95 06800 and 95 13570 and especially isoxaflutole, a selective maize herbicide, diketonitriles such as those described in European Applications 0 496 630, 0 496 631, in particular 2-cyano-3-cyclopropyl-1- (2-SO
2
CH
3
-4-CF
3
-phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO
2
CH
3
-4-2,3-Cl
2
-phenyl)propane-1,3-dione, triketones described in European Applications 0 625 505 and 0 625 508, in particular sulcotrione. However, a tolerance gene to such herbicides has not been described.
Hydroxyphenylpyruvate dioxygenase is an enzyme which catalyses the conversion reaction of para-hydroxyphenylpyruvate into homogentisate.
In addition, the amino-acid sequence of hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 has been described, without there being a description of its role in the tolerance of the plants to herbicides (Rüetschi et al.: Eur. J. Biochem. 205, 459-466, 1992). This document does not give a description of the gene coding for this protein.
There have now been discovered the sequence of a gene of this type and that such a gene can, once incorporated into plant cells, produce an over-expression or an activation of HPPD in the plants giving to the latter an worthwhile tolerance to certain novel herbicides, such as those of the isoxazoles family or that of the triketones.
An object of the present invention is an isolated DNA sequence of a gene of non-human origin of a non-marine bacterium, or alternatively of a plant gene, or a sequence which can hybridize with this sequence, characterized in that it expresses a hydroxyphenylpyruvate dioxygenase (HPPD).
More particularly, this sequence can be of bacterial origin, such as especially the genus Pseudomonas or alternatively of plant origin, such as especially of monocotyledonous or dicotyledonous plants, especially of Arabidopsis or of Umbelliferae, such as, for example, the carrot (
Daucus carotta
). It can be native or wild or possibly mutated while at the same time fundamentally retaining a property of herbicidal tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family or that of the triketones.
The invention likewise relates to a process of isolating the above gene, characterized in that:
as primers, some oligonucleotides from the amino-acid sequence of an HPPD are chosen,
starting from these primers, amplification fragments are synthesized by PCR
the gene is isolated by creation and screening of a genomic bank and
the gene is cloned.
Preferably, primers from the HPPD sequence of a bacterium of the genus Pseudomonas is used. Particularly preferably, they are from
Poeudomonas fluorescens.
The invention also relates to the use of a gene coding for HPPD in a process for the transformation of plants, as a marker gene or as a coding sequence allowing tolerance to certain herbicides to be conferred on the plant. It can likewise be used, in association with other marker genes and/or coding sequences, for an agronomic property.
The coding gene can be of any origin, native or wild or possibly mutated, while at the same time fundamentally retaining a property of herbicidal tolerance against inhibitors of HPPD, such as herbicides of the isoxazoles family or that of the triketones. As coding sequence, especially that described above can be used.
The transformation of plant cells can be achieved by any appropriate known means. A series of methods consists in bombarding cells or protoplasts with particles to which are coupled the DNA sequences.
Another series of methods consists in using, as means of transfer into the plant, a chimeric gene inserted into a Ti plasmid of
Agrobacterium tumefaciens
or Ri plasmid of
Agrobacterium rhizogenes.
An object of the present invention is also a chimeric gene comprising, in the transcription direction, at least one promoter regulation sequence, a heterologous coding sequence which expresses hydroxyphenylpyruvate dioxygenase and at least one terminator or polyadenylation regulation sequence.
The promoter regulation sequence used can be any promoter sequence of a gene which 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 biscarboxylase (RuBisCo) or that of a gene of &agr;-tubulin (European Application EP No. 0 652 286), or alternatively of a plant virus gene such as, for example, that of cauliflower mosaic virus (CAMV 19S or 35S), but any suitable promoter can be used. Preferably, recourse is made to a promoter regulation sequence which favours the overexpression of the coding sequence, such as, for example, that comprising at least one histone promoter such as described in European Application EP 0507698.
According to the invention, it is equally possible to use, in association with the promoter regulation sequence, other regulation sequences which are situated between the promoter and the coding sequence, such as “enhancer” transcription activators, such as, for example, tobacco etch virus (TEV) translation activator described in the Application WO87/07644, or of transit peptides, either single, or double, and in this case possibly separated by an intermediate sequence, that is to say comprising, in the transcription direction, a sequence coding for a transit peptide of a plant gene coding for a plastid localization enzyme, a part of the sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localization enzyme, formed by a part of the sequence of the N-terminal mature part of a plant gene coding for a plastid localization enzyme, such as described in European Application No. 0 508 909.
It is possible to use as terminator or polyadenylation regulation sequence any corresponding sequence of bacterial origin, such as, for example, the nos terminator of
Agrobacterium tumefaciens,
or even of plant origin, such as, for example, a histone terminator such as described in European Application EP No. 0 633 317.
An object of the present invention is also plant cells, of monocotyledonous or dicotyledonous plants, especially of crops, transformed according to one of the processes described above and comprising in their genome an efficacious quantity of a, gene expressing hydroxyphenylpyruvate dioxygenase (HPPD). It has been observed that transformed plants of this type have a significant tolerance to certain novel herbicides such as the isoxazoles described especially in French Patent Applications 9506800 and 95 13570 and especially of 4-[4-CF
3
-2-(methylsulphonyl)benzoyl]-5-cyclopropylisoxazole, and especially isoxaflutole, a selective maize herbicide, the diketonitriles such as those described in European Applications 0 496 630, 0 496 631, in particular 2-cyano-3-cyclopropyl-1-(2-SO
2
CH
3
-4-CF
3
-phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO
2
CH
3
-4-2,3-Cl
2
-phenyl)propane-1,3-dione, the triketones described in European Applications 0 625 505 and 0 625 508, in particular sulcotrione.
Finally, an object of the invention is a method of weeding plants, especially crops, with the aid of a herbicide of this type, characterized in that this herbicide is applied to plants transformed according to the invention, both pre-sowing, pre-emergence and post-emergence of the crop.
An object of the invention is also the use of the HPPD gene as a marker gene in the course of the “transformation-regeneration” cycle of a plant species and selection on the above herbicide.
REFERENCES:
patent: 5635618 (1997-06-01), Capellades
patent: 0507698 A1 (1992-10-01), None
patent: 0508909 A1 (1992-10-01), None
patent: 0614970 A2 (1994-02-01), None
patent: 0652286 A1 (1995-05-01), None
patent: WO 97/2
Matringe Michel
Pallett Ken
Rolland Anne
Sailland Alain
Aventis Cropscience S.A.
Benzion Gary
Connolly Bove & Lodge & Hutz LLP
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