Etude par génétique inverse du gène codant la protéine TARGET OF RAPAMYCIN d'Arabidopsis thaliana (AtTOR), l'homologue d'une kinase contrôlant la croissance cellulaire chez les eucaryotes

Benoit Menand 1
1 LGBP - Luminy Génétique et Biophysique des Plantes
BIAM - Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) : DRF/BIAM
Abstract : TOR (target of rapamycin) protein kinases were identified in yeast, mammals and Drosophila as central controllers of cell growth. Thu, G1 to S phases progression through the cell cycle is blocked by rapamycin, a drug which specifically inhibits TOR activity by forming a ternary complex with the peptidyl-prolyl isomerase FKBP12 (FK506 and rapamycin binding protein), and the FKBP-rapamycin binding domain (FRB) of TOR proteins. This work presents the study, the Arabidopsis homologue of yeast and animals TOR genes. By using RT-PCR and 5'-RACE, we cloned the cDNA of the single Arabidopis TOR (AtTOR) gene which contains 55 introns and encodes a protein of 300 kDA showing a high level of identity with its homologues from yeast and animals. However, the vegetative growth of Arabidopsis and other plants tested is insensitive to rapamycin. Nonetheless, we have shown, using a two hybrid system, that the AtTOR FRB domain is able to bind yeast FKBP12 in the presence of rapamycin. The function of AtTOR has been studied by reverse genetics. Two mutants, tor-1 and tor-2, have been identified in the INRA T-DNA insertion library (Versailles, France). In both mutants, the T-DNA is inserted upstream of the FRB and kinase domains of AtTOR. The two mutants do not complement each other and have an embryo-lethal phenotype in which one quarter of the seeds from a selfed pollinization of a heterozygotic parent die because of arrested development of the embryo and endosperm. We used a translational fusion of AtTOR with the GUS reporter gene in the tor-1 mutant to show that AtTOR expression is restricted to the developing endosperm and embryo, and all the primary meristems of the plant. This is different to mammalian and Drosophila TORs, which are expressed in all tissues. Theses results allow us to discuss the role of AtTOR in cell growth and proliferation Furthermore, experiments attempting to render Arabidopis sensitive to rapamycin, by transformation with yeast FKBP12, were undertaken and constructs for AtTOR overexpression using the GAL4 system were made.
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Submitted on : Tuesday, October 6, 2009 - 9:09:36 PM
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Benoit Menand. Etude par génétique inverse du gène codant la protéine TARGET OF RAPAMYCIN d'Arabidopsis thaliana (AtTOR), l'homologue d'une kinase contrôlant la croissance cellulaire chez les eucaryotes. Biologie végétale. Université de Strasbourg, 2002. Français. ⟨tel-00422428⟩

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