Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-07-18
2001-11-06
Benzion, Gary (Department: 1649)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023600, C536S023200, C800S288000, C800S300000, C800S287000
Reexamination Certificate
active
06313282
ABSTRACT:
The present invention relates to the use of terminator regions isolated from plant transcribed genes, of new chimeric genes containing them and their use for the transformation of plants.
Numerous phenotypic characteristics associated with the expression of one or few gene elements can be integrated into the genome of plants and thus confer advantageous agronomic properties on these transgenic plants. In a non-extensive manner, there may be mentioned: the resistance to pathogenic agents for crops, the resistance to phytotoxic plant-protection products, the production of substances of food or pharmacological interest. In addition to the isolation and characterization of the gene elements encoding these various characters, an appropriate expression should be provided. This appropriate expression can be situated both at the qualitative and quantitative level. At the qualitative level, for example spacial level: preferential expression in a specific tissue, or temporal level: inducible expression. At the quantitative level, by the accumulated quantity of the product of expression of the gene introduced. This appropriate expression depends, for a large part, on the presence of regulator gene elements associated with transgenes, in particular as regards the quantitative and qualitative elements. Among the primordial elements providing this appropriate regulation, the use of homologous or heterologous, single or combined promoter regions has been widely described in the scientific literature. The use of terminator region downstream of the transgene has been used for the sole purpose of putting a boundary which makes it possible to stop the transgene transcription process, without presupposition as to their role in the quality or quantity of the expression of the transgene.
The present invention relates to the use of terminator regions isolated from plant transcribed genes, of new chimeric genes containing them and their use for the transformation of plants. It relates more particularly to the simultaneous use of terminator regions and promoters isolated from the same plant transcribed gene. It permits the appropriate expression, both quantitative and qualitative, of the transgenes under the control of these gene regulatory elements. This appropriate expression obtained by the use of the present invention may relate to characters such as: the resistance to pathogenic agents for crops, the resistance to phytotoxic plant-protection products and the production of substances of food or pharmacological interest. In particular, it makes it possible to confer an increased herbicidal tolerance on the transgenic plants by a preferential, qualitative and quantitative expression of the product of expression of the chimeric genes in the regions of the plant undergoing rapid growth. This specific appropriate expression of the gene for herbicidal resistance is obtained by the simultaneous use of the promoter and terminator region regulatory elements of the H4A748 histone gene from
Arabidopsis thaliana.
Such an expression pattern can be obtained for all the characters of interest, as described above, with the regulator elements used to confer an increased herbicidal tolerance. The present invention also relates to the plant cells transformed by means of these genes and the transformed plants regenerated from these cells as well as the plants derived from crossings using these transformed plants.
Among the plant-protection products used for the protection of crops, systemic products are characterized in that they are transported in the plant after application and, for some of them, accumulate in the parts undergoing rapid growth, especially the stem and root apexes, causing, in the case of herbicides, the deterioration, up to the destruction, of the sensitive plants. For some of the herbicides exhibiting this type of behaviour, the primary mode of action is known and results from an inactivation of characterized enzymes involved in the pathways of biosynthesis of compounds necessary for the correct development of the target plants. The target enzymes for these products can be located in various subcellar compartments and the observation of the mode of action of known products most often shows a localization in the plastidial compartment.
The tolerance of plants sensitive to a product belonging to this group of herbicides, and whose primary target is known, can be obtained by stable introduction into their genome of a gene encoding the target enzyme, of any phylogenetic origin, mutated or not as regards the characteristics of inhibition, by the herbicide, of the product of expression of this gene. Another approach consists in stably introducing into the genome of the sensitive plants a gene of any phylogenetic origin encoding an enzyme capable of metabolizing the herbicide into an inactive and non-toxic compound for the development of the plant. In this latter case, it is not necessary to have characterized the target of the herbicide.
Given the mode of distribution and accumulation of products of this type in the treated plants, it is advantageous to be able to express the product of translation of these genes so as to permit their preferential expression and accumulation in the regions of the plant undergoing rapid growth where these products accumulate. Furthermore, in the case where the target of these products is localized in a cellular compartment other than the cytoplasm, it is advantageous to be able to express the product of translation of these genes in the form of a precursor containing a polypeptide sequence permitting the tolerance-conferring protein to be addressed to the appropriate compartment, and in particular to the plastidial compartment.
As example illustration this approach, there may be mentioned glyphosate, sulphosate or fosametin which are broad-spectrum systemic herbicides of the phosphonomethylglycine family. They act essentially as competitive inhibitors, with respect to PEP (phosphoenolpyruvate), of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19). After their application of the plant, they are transported into the plant where they accumulate in the parts undergoing rapid growth, especially the stem and root apexes, causing the deterioration, up to the destruction, of the sensitive plants.
EPSPS, the main target of these products is an enzyme of the pathway of aromatic amino acid biosynthesis which is localized in the plastidial compartment. This enzyme is encoded by one or more nuclear genes and synthesized in the form of a cytoplasmic precursor and then imported into the plastids where it accumulate in its mature form.
The tolerance of the plants to glyphosate and to products of the family is obtained by the stable introduction into their genome of an EPSPS gene of plant or bacterial origin, mutated or not as regards the characteristics of glyphosate inhibition of the product of this gene. Given the mode of action of glyphosate, it is advantageous to be able to express the product of translation of this gene so as to permit its high accumulation in the plastids and furthermore, in the regions of the plant undergoing rapid growth where the products accumulate.
It is known, for example according to U.S. Pat. No. 4,535,060, to confer on a plant a tolerance to a herbicide of the above type, in particular, N-phosphonomethylglycine or glyphosate, by introduction into the genome of the plants of a gene encoding an EPSPS carrying at least one mutation rendering this enzyme more resistant to its competitive inhibitor (glyphosate), after localization of the enzyme in the plastidial compartment. These techniques however need to be improved for a greater reliability in the use of these plants during a treatment by these products under agronomic conditions.
In the present description, “plant” is understood to mean any differentiated multicellular organism capable of photosynthesis and “plant cell” is understood to mean any cell derived from a plant and capable of constituting undifferentiated tissue such as calli, or differentiated tissue such as embryos or plant p
Atanassova Rossitza
De Rose Richard
Freyssinet Georges
Gigot Claude
Lebrun Michel
Benzion Gary
Rhone-Poulenc Agrochimie
Scully Scott Murphy & Presser
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