Cloning And Preliminary Funciton Analysis Of A TOR Gene From Brachypodium Distachyon

TOR (Target of Rapamycin) is a evolutionary conservative Ser/Thr kinase in eukaryotes, whichbelongs to phosphatidylinositol3-kinase-related protein kinases (PIKK) family. A macrolideimmunosuppressant rapamycin binds FKBP12and inhibits TOR. In diverse eukaryotes, TOR is acentral regulator of cell growth, metabolic, nutrients, energy, growth factors and stress responses. TORconsist of highly conserved domains, they are HEAT repeats, FAT domain, FRB domain, kinase domain,FATC domain from N-terminal to C-terminal. In yeast and animals, TOR form two different complexes,TOR complex1(TORC1) and TOR complex2(TORC2). TORC1regulates translation,ribosomebiogenesis and cell growth in response to the change of nutrients and energy, and it is rapamycinsensitive. TORC2regulates cytoskeleton,cell cycle and apoptosis,and it is not inhibited by rapamycin.Current research about plants TOR is focus on Arabidopsis, and little is known about plants TOR. WTArabidopsis is rapamycin insensitive, but the transgenic Arabidopsis BP12, overexpression ofScFKBP12, is rapamycin sensitive, it is growth inhibition with rapamycin treatment.A TOR gene is cloned from Brachypodium distachyon in this study, and following conclusions aredraw from preliminary analysis its structure and function.1.Conventional dose (1μM) of rapamycin have no effect on germination and growth ofBrachypodium, but high concentration (100μM) of rapamycin have significant inhibition effect.2.A7401bp gene named BdTOR is cloned from Brachypodium distachyon. BdTOR codes a2466aaprotein, and domain projection suggest that it has HEAT repeats(1-1296), FAT domain (1297-1877),FRB domain (1755-2110), kinase domain (2077-2466), FATC domain (2434-2466).3.Real-Time PCR showed that the relative expression of BdTOR in Brachypodium from highest tolowest are green seed, young seedling, root, leave and stem.4.Transient expression revealed BdTOR kinase domain (2077-2466) located in cell nuclear andcytoplasmic ofArabidopsis protoplasts, and it is compartmentalization in cytoplasmic.5.Construction of plant expression vectors pOEBdTOR, pOEBdTORF1, pOEBdTORF2,pOEBdTORF3for transform WT and BP12. Overexpression of BdTOR (1-2466)、BdTORF1(1-1259)、BdTORF2(1251-2466)、BdTORF3(1745-2466) to study their function.6.Overexpression of BdTOR or its fragment in BP12can recover or part recover the inhibition byrapamycin, but do not change the character of rapamycin sensitive. It revealed a possibility ofinteraction between FRB of BdTOR and ScFKBP12in the treatment of rapamycin.7.Overexpression of BdTOR in BP12or WT can improve size of leaf, length of main root,development of root, and enable greater biomass of Arabidopsis. It means that overexpression ofBdTOR in Arabidopsis can improve its growth rate.8.Sucrose can improve the growth of Arabidopsis, and overexpression of BdTOR enhanced theeffect. It revealed the closely relationship of sugar metabolism, plant growth and TOR function.In conclusion, this study first cloned BdTOR from Brachypodium distachyon, and preliminary analysis its structure and function. It provides a basis for further studies on the biological function ofBdTOR, ant it also provide a new alternative gene for molecular breeding.