T20N点突变对水稻OsRacD蛋白GTP酶活性的影响

Effects of T20N Site-directed Mutation on GTPase Activities of OsRacD from Oryza sativa

OsRacD是水稻小GTP结合蛋白Rho家族成员,其功能之一是作为"分子开关",通过控制花粉管的延伸生长,参与光敏核不育水稻光周期的育性转换。在序列同源性比对和蛋白保守结构域分析的基础上,采用重叠延伸PCR方法在水稻OsRacD基因的第一个高度保守的基序G1区引入T20N点突变,模拟GDP结合形式的OsRacD。进一步构建了与组氨酸标签融合的原核表达载体,原核表达和纯化了野生型和突变型OsRacD蛋白,并通过Western blot证实了融合蛋白表达和纯化的正确性。纯化蛋白的GTP酶活性检测结果显示,突变后的OsRacD蛋白GTP水解活性显著降低,提示OsRacD在T20N突变前后具有不同的生化特性。

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.