水稻OsHsfA7基因RNA干扰载体的构建及遗传转化研究

CONSTRUCTION OF RNAi EXPRESSION VECTOR AND TRANSFORMATION OF OSHSFA7 GENE IN Oryza sativa L.

为研究水稻热激转录因子基因OsHsfA7的功能及其在水稻耐热育种方面的应用价值,本文通过构建水稻OsHsfA7基因RNA干扰载体获得功能抑制变异材料,从反向遗传学进行基因的功能分析。扩增OsHsfA7c DNA3'编码区470bp片段,分别以反向和正向插入到中间载体pBSK连接的GUS片段两侧,然后将得到的RNA干扰片段克隆到改造的植物表达载体p1301M,构建以CaMV35S启动子驱动表达的水稻OsHsfA7基因RNA干扰表达载体。将该载体转入根癌农杆菌EHA105后,采用农杆菌介导法进行了水稻日本晴品种的遗传转化,获得了23株具有潮霉素抗性的转基因植株,其中12株经GUS染色呈蓝色并且DNA检测10株已插入目的片段,RNA检测其中6株OsHsfA7基因的表达水平下降甚至未检出。结果说明本研究构建的OsHsfA7基因RNA干扰载体对该基因表达沉默是有效的。

RNAi expression vector of rice heat shock transcription factor OsHsfA7 was constructed and transformed into Oryza sativa L.to generate low or null expression mutants for the functional characterization by reverse genetics and evaluate its application value on rice breeding for heat tolerance.A 470bp fragment amplified from the 3 ＇end of OsHsfA7 cDNA were inserted into both sides of GUS fragment linked with intermediate vector pBSK in reverse and forward direction,respectively.The RNA interference fragment was then cloned into a reconstructive vector p1301M to form OsHsfA7 RNA interference expression vector driven by CaMV35S promoter.The recombinant was transformed into Oryza sativa L.mediated by Agrobacteria tumefaciens EHA105.Twenty-three transgenic rice plants were obtained by hygromycin resistance selection and twelve of the transformant leaves showed blue by GUS staining.Successful intergration of the RNA interference fragment was confirmed in ten transgenic plants by DNA amplification and RT-PCR amplification showed that the transcript levels of OsHsfA7 in six transgenic plants were greatly decreased or undetectable.The results demonstrated that the OsHsfA7 RNA interference vector was effective for this gene silencing in Oryza sativa L.