烟草orf25基因RNAi表达载体的构建及遗传转化

Construction of orf25 Gene RNA Interference Expression Vector and Transformation of Tobacco

杂种优势的利用可以有效地实现烟草高产、高抗、优质的育种目标,目前,培育雄性不育系是获取杂种烟草最有效的途径。研究发现orf25基因位于线粒体基因组中,与烟草雄性不育密切相关。本研究以烟草雄性不育相关基因orf25为研究对象,根据RNA干扰载体构建原则,将长度为606 bp的orf25基因干扰片段分别以正向、反向连接到大小为190 bp的linker连接片段的两侧,构成发夹结构的RNA干扰载体片段。将RNA干扰载体片段插入到中间载体pET30a上,最后克隆到植物表达载体pCAMBIA1301中,成功构建orf25基因的RNAi表达载体。利用农杆菌介导法将pCAMBIA1301-orf25-RNAi转入到烟草保持系中烟90中,经抗性筛选以及分子检测最终获得35株转pCAMBIA1301-orf25-RNAi烟草植株,其中阳性烟草有18株,转化率为51.4%。结果表明pCAMBIA1301-orf25-RNAi干扰载体已成功转入到烟草中烟90中。

The utilization of heterosis can effectively make the tobacco achieve high yield, high resistance and high quality breeding target. At present, breeding male sterile lines is the most effective way to get hybrid tobacco. Studies have found that the off25 gene is located in the mitochondrial genome, which is closely related to the male sterility of tobacco. With the research object of the tobacco male sterility related genes orf25, based on the RNAi expression vector construction principle, off25 forward and reverse siRNA fragment with length of 606 bp were inserted into both sides of linker with length of 190 bp to constitute RNA interference fragment with stable hairpin structure. Then the RNA interference vector was inserte d into the intermediate vector pET30a. Finally, it was cloned into the plant expression vector pCAMBIA1301. RNAi expression vector with orf25 gene was successfully constructed. We transferred pCAMBIA1301-orf25-RNAi into tobacco male-fertile varieties Zhongyan90 by agrobacterium mediated method. 35 transgenic pCAMBIA 1301- orf25-RNAi tobacco plants were obtained. PCR indentification proved that transgenic positive plants were 18, the conversion rate was 51.4%. The results showed that the pCAMBIA1301-orf25-RNAi interference vector had been successfully transferred to tobacco male-fertile varieties Zhongyan90.