Exogenous DsRNA Interference Sugarcane Mosaic Virus Resistance Regulation

Maize(Zea mays L.) is one of the most important crops, staple food, forgage crop and industry materials. However viral diseases, particularly maize dwarf mosaic disease caused by Sugarcane mosaic virus and maize rough dwarf disease are among the most damaging diseases affecting maize production and quality in China. It is urgency to cultivate and popularize disease-resistant cultivar and search for new methods. RNA interference (RNAi), as an important RNA-based reverse-genetic system, has been used for gene function and genetic melioration studies in plants. RNAi has displayed great advantage in antiviral of breeding which has been proved to be a technology distinctiveness, high efficiency, economy, and inhibiting gene expression, and has become the focus of scientist researches. In order to select and establish the prokaryotic expression system of the cp gene from virus, we explored a basic principle of inverted-repeat expression vector design, and report an efficient method to produce dsRNA using a bacterial expression system. Our work illustrates some basic principles for protecting plants against viral diseases in vitro.The main results are summarized as follow:1. According to cp gene sequence of sugarcane mosaic virus, specific primers were designed,147bp upstream and140bp downstream fragments were amplified by RT-PCR.2. Based on the cloning vector pUCCRNAi, Two inverted repeat stem-loop vector pUCCRNAi+2F were constructed, and then digested with Pst I-Sal I, and inserted into the prokaryotic expression vector L4440, two expressed vectors LMCP1and LMCP2were constructed.3. The two recombinant plasmids were transformed individually into E.coli HT115, and dsRNA was induced by IPTG. The results showed that the expression products were the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of0.4-0.6mmol/L and induction time of4h was the most optimal expression condition.4. The crude extracts of E.coli HT115containing large amounts of dsRNA were sprayed to plants and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (cpl) was more effective than was dsRNA derived from a downstream region (cp2) of the SCMV cp gene. 5. The results showed that plants inoculated with the virus and LMCP preparation, diluted1/5or less, and treated at or before5days prior to viral inoculation, displayed decreased disease symptoms. Besides, dsRNA-containing extracts with penetrating agent of suitable selection concentration could Inhibit virus infection effectively.In summary, maize inbred line8112was inoculated with crude extracts of bacterially expressed dsRNAs to protect maize plants against SCMV infection and viral infection was detected. RNAi technique system of dsRNA in vitro was filtered and constructed. The results provide a valuable tool for plant viral control using dsRNA and the PTGS approach.