OsVIP1 RNAi转基因水稻植株的构建

Construction of OsVIP1 RNAi transgenic rice plants

为进一步阐明农杆菌介导的遗传转化分子机制,进而利用分子生物学方法提高农杆菌转化水稻效率,以水稻品种中花11为遗传转化受体材料,利用RNAi技术对水稻中拟南芥AtVIP1蛋白的同源蛋白进行了功能研究。通过同源性比对得到水稻中拟南芥AtVIP1蛋白的同源蛋白序列,命名为OsVIP1,构建了该蛋白基因2个片段(R1和R2)的RNAi表达载体pDS1301-2-R1和pDS1301-2-R2,通过农杆菌介导的方法分别转化水稻中花11。对转基因抗性愈伤进行统计,结果表明,由携带pDS1301-2-R1和pDS1301-2-R2载体转化的抗性愈伤的形成受到一定程度抑制。经PCR检测,证明2个片段R1和R2均成功整合到再生水稻植株基因组中;半定量RT-PCR分析显示部分RNAi转基因植株中OsVIP1基因表达被成功抑制。

In order to elucidate the molecular mechanism of Agrobacterium-mediated genetic trans- formation and improve the efficiency of Agrobacterium-mediated rice transformation by molecular biological methods,we studied the OsVIP1 protein function with the RNA interfering technology using the rice variety Zhonghua 11 as the host for genetic transformation. A rice protein sequence homologous to AtVIP1, named OsVIP1 was obtained by homologous searches. Two RNAi expression vectors, pDS1301- 2-R1 and pDS1301-2-R2,were constructed with two fragments （R1 and R2） of the OsVIP1 cDNA, respectively, and used to transform rice variety Zhonghua 11 callus through Agrobacterium. The statistical results showed that formation of the transgenic resistant callus of pDS1301-2-R1 and pDS1301-2-R2 vectors was inhibited to some extent. PCR experiments indicated that R1 and R2 fragments were successfully integrated into the transgenic rice genome. Semi-quantitative RT-PCR analysis showed that the OsVIP1 gene expression was suppressed successfully in some RNAi transgenic rice plants.