Study On The Protein-protein Interaction Between OsRhoGDI2and OsRacD In Rice

OsRacD belonging to Rho family of the small GTP binding proteins in rice was separated fromyoung panicles of the rice at the stage of pistil and stamen formation.The previous studies showed that itwas a very important gene on regulation of the rice growth and development by influencing rice fertility.Using OsRacD as bait,the rice panicle cDNA library was screened by yeast two-hybrid system,and somecDNA sequences encoding putative targets were obtained,one of the which is a positive clone namedOsRhoGDI2(AY364312).In this paper, we focus on the protein-protein interaction between OsRhoGDI2and OsRacD.The expression of OsRhoGDI2and OsRacD genes was analyzed by the RT-PCR during the ricegrowth periods.Results showed that they expressed at each stage,and in young panicles of rice,theyexpressed highest of all.The rule of their expression characters was similar.Results demonstrated that theymight cooperative control the rice fertility.To study their interaction,the wild type,constitutively activated and inactived OsRacD withOsRhoGDI2were tested in the yeast two-hybrid system.and the results revealed that wild type andconstitutively activated OsRacD can both bind with OsRhoGDI2,which indicated that their interaction wereindependent of the active state of OsRacD. In order to determine their interaction domain between OsRacDand OsRhoGDI2, PCR-mediated method was used for deletion mutation,and the effects were detected bythe yeast two-hybrid system. The results showed that only by the conserved domain itself can not facilitatethe binding between OsRhoGDI2and OsRacD.In order to identify their binding relationship invitro,GST-fused OsRhoGDI2, GST-fused the the deletion mutant of OsRhoGDI2and His6tag-fusedOsRacD were expressed and purified from E. coli.The GST pull down results showed the same rule.To investigate their interaction in vivo with bimolecular fluorescence complementation (BiFC)method,OsRhoGDI2was fused with the C-terminal domain of the yellow fluorescentprotein(YFP),OsRacD and G15V-OsRacD was fused with N-terminal domain of YFP.And the transientexpression vectors which were named as pUC-SPYCE-OsRhoGDI2,pUC-SPYNE-OsRacD andpUC-SPYNE-G15V-OsRacD were constructed and then delivered into the cell of the leaf epidermis of tobacco.The fluorescent signals appeared in the nucleus,which showed that they actually interacted there.In this study, the interaction between OsRacD and OsRhoGDI2were analyzed through biochemical,molecular biology and cytology methods.Their relationship in rice development processes help to clarifyand reveal the molecular mechanism of rice photoperiod fertility conversion from the aspect ofprotein-protein interaction in signalling pathways.