细菌表达dsRNA介导的家蚕FTZ-F1基因的RNA干扰

为探索细菌表达目标基因dsRNA介导的RNAi技术是否在家蚕Bombyx mori可行, 本研究引入了在其他物种中广泛应用的细菌表达dsRNA的RNAi系统: HT115细菌株和L4440质粒。利用L4440载体两端含有T7启动子的特点, 设计并构建了针对家蚕核受体FTZ-F1基因的RNA干扰（RNA interference）载体, 将构建好的质粒转入大肠杆菌Escherichia coli HT115, 在IPTG诱导下成功获得目标基因对应双链RNA（dsRNA）。 结果显示: 通过对5龄第7天家蚕幼虫注射IPTG诱导后提取的FTZ F1基因对应的dsRNA 25 μg, 85%的蛹变态发育过程明显延迟, 不能实现幼虫到蛹的形态完全转变。荧光定量PCR分析显示目标基因的表达得到了特异的抑制。实验结果初步表明, 通过细菌表达目标基因dsRNA介导的RNAi策略, 以其经济、高效的特点, 具有广泛应用于家蚕基因功能研究中的潜力。

RNA interference of FTZ-F1 gene mediated by bacterially expressed dsRNA in the silkworm, Bombyx mori

We developed a method of RNA interference based on bacterially expressed dsRNA in the silkworm, Bombyx mori. By inserting the target, FTZ-F1 gene fragment, between the two convergent T7 polymerase promoters in opposite orientation in L4440 dsRNA expression vector, the recombinant plasmid was formed. Then the recombinant plasmid was transformed into Escherichia coli HT115, an RNase-III deficient strain. dsRNA was extracted from the E. coli HT115 after being treated with isopropyl-β-D-thio-galactopyranoside (IPTG). RNAi treatment was performed by injecting the extracted dsRNA (25 μg) into body cavity of silkworm at the 7th day of 5th instar. The RNAi of FTZ-F1 gene resulted in 85% of the insects with delay of pupal metamorphosis and disablement in pupa formation. Real-time quantitative PCR analysis revealed that the expression of FTZ F1 gene was specially inhibited after the insects were treated with dsRNA of FTZ-F1 gene. The results suggest that the bacterially expressed dsRNA has potential to be used in silkworm functional genome analysis in an economical and efficient way.