利用Red重组系统敲除大肠杆菌rnc基因构建dsRNA原核表达体系

KNOCK-OUT THE RNC GENE OF E COLI USING RED-MEDIATED RECOMBINATION FOR CONSTRUCTION OF DSRNA PROKARYOTIC EXPRESSION SYSTEM

大肠杆菌的rnc基因编码产物为RNaseIII酶,RNaseIII酶能降解细菌中绝大多数dsRNA。利用来源于λ噬菌体的Red重组系统和重叠延伸PCR技术(gene splicing by overlap extension PCR,SOE-PCR),敲除了大肠杆菌origami(DE3)菌株的rnc基因,获得了RNaseIII缺失型菌株M-origami。利用电激法,将构建的TMV运动蛋白基因(movement protein gene,MP)的dsRNA表达载体LMP480导入M-origami菌株中,IPTG诱导表达的结果显示:构建的M-origami/LMP480原核表达系统能高效表达TMV运动蛋白基因的dsRNA。初步的抗病性鉴定显示,表达的dsRNA能够诱发烟草对TMV的抗性。

The rnc gene of E. coli can encode the RNaseⅢ protein which can degrade most of dsRNA transcripts in E. coll. The rnc gene of origami （ DE3 ） strain using λ - dependent Red - mediated recombination system and gene splicing by overlap extension PCR and the M -origami strain deficient for RNaseⅢ. wat And achieved the vector of LMP480 that can express TMV MP dsRNA transcripts was amstueted and transformed it into M - origami strain by electroporation. Induced by IPTG, M -origami/LMP480 strains could effectively express TMV MP dsRNA transcripts and the dsRNA could protect tobacco from the infection of TMV.