摘要
[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。
[ 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 transformd 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 HTll5 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.
作者
王雪庆
赵遵岭
孙涛
陈晓鸣
杨璞
WANG Xue-qing;ZHAO Zun-ling;SUN Tao;CHEN Xiao-ming;YANG Pu(Resources Insects,Chinese Academy of Forestry,Key Laboratory of Cultivating and Utilization of Resources Insects of State Forestry Administration,Kumning 650224,Yunnan,Chin)
出处
《林业科学研究》
CSCD
北大核心
2018年第4期70-74,共5页
Forest Research
基金
林业公益性行业科研专项201504302
国家自然科学基金31572337
中央级科研院所基金CAFYBB2017ZB005
