白蜡虫ws基因RNAi载体构建及原核表达dsRNA

目的]构建白蜡虫(Ericerus pela)蜡酯合酶(wax synthase,WS)基因干扰载体并建立其体外dsRNA(doublestranded RNA,dsRNA)原核表达体系,低成本大量制备白蜡虫ws基因的dsRNA。方法]克隆白蜡虫蜡酯合酶基因ws片段,连入L4440载体,将重组质粒转入大肠杆菌HT115感受态细胞,经IPTG诱导获得与目的片段相对应的dsRNA。结果]白蜡虫ws基因RNA干扰(RNA interference,RNAi)载体成功构建,重组质粒转入HT115感受态细胞经IPTG诱导后菌体成功表达dsRNA,dsRNA的平均获得量1 705 ng·m L~(-1)。结论]该研究通过原核表达白蜡虫ws基因的dsRNA,为后续利用RNAi实验研究白蜡虫ws基因功能及作用机理奠定基础。

Construction of RNA Interference Vector of Ericerus pela Chavannes) ws Gene and Preparation of dsRNA by Prokaryotic Expression

Objective] This study aims at construct the interference vector of Ericerus pela wax synthase gene and prokaryotic expression system in vitro, and prepare a large number of double-stranded RNA (dsRNA) of E. pela ws gene at low cost.Method] The cloned E. pela ws gene fragment was inserted into L4440 vector to construct E. pela ws-L4440 gene interference vector. The recombinant plasmid was transformed into HT115 competent cell, then induced by IPTG to get the dsRNA corresponding to target fragment.Result] The interference vector of Ericerus pela ws gene was successfully constructed in vitro, and the dsRNA can also be expressed by HT115 competent cell with transformed recombinant plasmid induced by IPTG. The average production of dsRNA was 1 705 ng·mL^-1.Conclusion] The expression of the dsRNA of E. pela ws gene by prokaryotic expression system may lay foundation for using RNAi technology to study the function and mechanism of E. pela ws gene.