玉米矮花叶病毒CP基因dsRNA的原核表达与分离

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Pst I-Sal I位点插入到L...

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

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.