The Construction of a Vector for Endosperm-specific Expression of IPT Gene and It's Transformation in Rice

Abstract

为研究细胞分裂素在水稻籽粒发育中的作用,构建了P1300-P Pg-5α-IPT-nOS植物表达载体,并对水稻进行遗传转化。以水稻品种9311为材料,采用PCr法获得种子中特异表达的Pg-5α基因启动子,并将此启动子与P1300相连接,构建P1300-P Pg-5α载体,用nCOⅠ和SPEⅠ双酶切P Sg516,获得IPT-nOS核酸片段,然后将此核酸片段插入到P1300-P Pg-5α中,构建P1300-P Pg-5α-IPT-nOS植物表达载体。以水稻品种日本晴愈伤组织为材料,采用农杆菌介导的方法进行遗传转化。成功构建了植物表达载体P1300-P Pg-5α-IPT-nOS,获得了阳性率为81.3%转IPT基因群体。获得转基因株系后,将为进一步筛选高效表达株系及观察其籽粒生长发育过程提供基础。In order to specifically express IPT gene in the endosperm of developing rice seed,the binary vector p1300-p PG-5α-IPT-Nos was constructed,firstly,the promoter of PG-5α was obtained by PCR amplification from rice genomic DNA( variety: 9311) and p1300-p PG-5α was constructed by inserting p PG-5α into p1300,then the IPT-Nos fragment was obtained by cutting p SG516 with Nco Ⅰ and Spe Ⅰ.After IPT-Nos inserted into p1300-p PG-5α,p1300-p PG-5α-IPT-Nos was constructed.p PG-5α-IPT-Nos was transformated into Oryza sativa( subsp.japonicacv.Nipponbare) cells by agrobacterium mediated method and regenerative seedlings were obtained.The results of PCR showed that about 81.3% seedlings were positive.唐山师范学院博士基金项目(09A01); 农业部“948”项目(2011-G1(3)-07); 河北省科技计划项目(13396401D