水稻叶绿体16S启动子克隆改造、载体构建及转化研究

Isolation and Modification of Rice Chloroplast 16S Promoter, Construction of Expression Vector and Transformation

利用PCR方法从水稻叶绿体基因组DNA中分离 1 6S启动子 ,并在其下游加入rbcL基因SD序列 ,以增强该启动子的翻译能力 ;序列分析表明 ,除加入的SD序列外 ,扩增片段与水稻 (Oryzasativa)叶绿体基因组DNA序列 1 6S启动子相应区域同源性为 1 0 0 %。将 1 6S启动子与bar基因和gfp基因的融合基因连接 ,以psbA基因的 3′序列为终止子 ,并以烟草叶绿体trnH_psbA和trnK为同源片段构建了烟草叶绿体表达载体pR1 6S。用基因枪转化烟草 ,转化植株经Southern、Northern检测及后代遗传学分析 ,发现1 6S启动子具有启动活性 。

Rice chloroplast 16S promoter was isolated by PCR from rice chloroplast genomic DNA. In order to enhance the ability of translation, SD sequence from rbcL gene was introduced into the downstream of the promoter. The sequence analysis showed that the isolated fragment and the rice( Oryza sativa ) chloroplast 16S promoter shared 100% homology except the introduced SD sequence. A Nicotiana tabacum chloroplast transformation vector pR16S has been constructed. It contains not only the chimeric gene bar and gfp gene regulated by 16S promoter and psbA teminator, but also two homologous fragments, trnH _ psbA and trnK . The plasmid pR16S has been transferred to Nicotiana tabacum by biolistic method. The transformants were identified by Southern_blotting,Northern_blotting and genetic analysis of progenies. The results showed that 16S promoter worked effectively and the foreign gene had been integrated in the chloroplast genome and inherited maternally to the progenies.