T20N点突变对水稻OsRacD蛋白GTP酶活性的影响

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。

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

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-OsRac...