Effects of T20N Site-directed Mutation on GTPase Activities of OsRacD from Oryza sativa / 中国生物工程杂志

ABSTRACT

OsRacD belonging to rice Rho family of the small GTP binding proteins,is a pivotal gene involved in rice photoperiod fertility conversion of photoperiod sensitive genic male sterile rice,which influences rice fertility via controlling the pollen tube growth.T20N site-directed mutation was introduced into its highly conserved G1 motif by PCR-mediated method to mimic its GDP-binding state based on the sequences alignment and conserved domains analysis.The prokaryotic expression vector of OsRacD and T20N-OsRacD were constructed,and the His6 tag fused proteins were expressed and purified from E.coli.After identified by Western blot,the GTP hydrolysis activities were detected.The results showed that the GTPase activities of T20N-OsRacD were significantly reduced comparing with that of OsRacD,suggested that OsRacD and T20N-OsRacD have different biochemical characteristics.