小麦AGPase基因LSU Ⅱ的克隆及反义表达与RNAi干扰载体的构建

Molecular Cloning of Plastidial Large Subunit Ⅱ Gene and Its Constructions of Antisense and RNAi Expression Vectors

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出小麦淀粉合成关键酶—AGPase质体型大亚基(AGPase plastidial large subunit,LSU Ⅱ)cDNA一段特异保守序列,长度为535 bp(GenBank No.EF378944),与DQ409820(小麦)、U66876(大麦)、AK069296(水稻)和DQ406819(玉米)分别有98%,97%,88%,88%的同源性,表明克隆出了小麦LSU Ⅱ基因的部分cDNA序列。以pWM101质粒表达载体为基础,将LSU Ⅱ反向置于pWM101质粒的CaMV35S启动子之后,构建了LSU Ⅱ的反义表达载体pWM101LSUIIA;另外,用pFGC5941干扰载体构建了LSU Ⅱ基因的RNAi载体pFGC5941 LSU ⅡSA,这些载体的构建为研究LSU Ⅱ基因的功能打下了基础。

Partial cDNA fragment （535 bp） of plastidial large subunit of AGPase （ LSU Ⅱ） from grains of common wheat variety Yujiao 2 was cloned （GenBank No. EF378944）. The sequence analysis demonstrated that the cloned LSU Ⅱ shared 88% -98% homogeneity with the sequences of DQ409820, U66876, AK069296, DQ406819 genes. The antisense expression vector （pWM101LSU ⅡA） was constructed with pWM101 plasmids, and RNAi vector （pFGC5941LSU IISA） was also constructed with pFGC5941. The constructed vectors provided a good background for studying the function of LSU Ⅱ.