玉米矮花叶病毒CP基因dsRNA的原核表达与分离

Prokaryotic Expression and Extraction of dsRNA Based on the CP Gene of Maize Dwarf Mosaic Virus

根据玉米矮花叶病毒CP基因序列设计特异性引物,RT-PCR扩增玉米矮花叶病毒CP基因特异性干涉片段,将干涉片段及pUCCRNAi载体分别用BamH I及Sal I双酶切,然后将干涉片段分别正反向插入pUC-CRNAi载体中,构建CP基因反向重复克隆载体pUCCRNAi+2 F。再利用PstⅠ-Sal I位点插入到L4440质粒中构建原核表达载体LMCP。利用IPTG进行诱导表达并对诱导表达条件进行优化。结果表明,经过IPTG诱导,LMCP在大肠杆菌HT115(DE3)菌株中可表达产生预期大小的核酸片段,经DNase I和RNase A消化处理,证实为dsRNA。同时IPTG浓度为0.4～0.6mmol/L,诱导表达4h,dsRNA的表达量最高。另外,溶解于ddH_2O中的dsRNA稳定性要高于溶解在NaCl中的,且随着放置时间的延长,dsRNA将出现明显的降解。

MDMV CP gene fragments were amplified by RT-PCR from extracted MDMV mRNA. To prepare a hairpin RNA, MDMV CP gene fragments and the pUCCRNAi cloning vector were digested by BamH I-Sal I respectively, First, the BarnH I-Sal I fragment from MDMV RNA was cloned in the positive orientation into pUCCRNAi to generate pUC- CRNAi ＋ F. And then, the other BamH I-Sal I fragment was cloned in the reverse orientation into Bgl Ⅱ-Xho I digested pUCCRNAi ＋ F to generate an inverted repeat sequence of pUCCRNAi ＋ 2 F （ sense orientation fragment and antisense o- rientation fragment were separated by an intron）. Thirdly, IA440 and pUCCRNAi ＋ 2 F plasmids were digested with Pst I-Sal I and subsequently joined to generate LMCP. And the recombinant plasmid was induced by IPTG. The results showed that the expression product was the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of 0.4 - 0.6 mmol/L and induction time of 4 h was the most optimal expression condition. The stability of the dsRNA in ddH20 is higher than that of in NaCl, and the dsRNA appeares to be dissolved with the time extending.