TNV-p82C-mediated Gene Silencing System And Artificially Designed MicroRNA Against Soybean Mosaic Virus

Gene silencing is a powerful tool to study the gene function of plant and the host factors required by viruses.Currently,gene silencing tools mainly include antisense RNA mediated gene silencing,co-suppression,RNAi and VIGS,each of which has its own advantages and disadvantages.We have developed a new virus-free gene silencing tool using RNA-dependent RNA polymerase(Rd Rp)of Tobacco necrosis virus A(TNV-A)in this study.Two vectors were overexpressed by agroinfiltration,a vector containing the C-terminal sequence of TNV-A Rd Rp,and another vector containing the promoter sequence responsible for the synthesis of TNV-A positive-sense RNA and the sequence of silenced gene,which could produce efficient gene silencing.We compared the gene silencing efficiency between this gene silencing system and Hairpin using phytoene desaturase(PDS)and secretion associated Ras1(SAR1)as the silencing target.The results showed that this system was more efficient than Hairpin.Besides,we have used this system to silence the gene of ER-to-Golgi transport related proteins with the infection of Soybean mosaic virus(SMV)in Nicotiana benthamiana.The accumulation of SMV coat protein(CP)was significantly inhibited when the gene of SAR1,SEC13,SEC31 or RAB1 was silenced,which suggests these proteins are the host factors of SMV.Overall,we proved that this gene silencing system is more effective than Hairpin,and it is a powerful tool to study the host factors of viruses.Soybean mosaic virus disease seriously affects the yield and quality of soybean in China.SMV is the main pathogen of soybean mosaic virus disease.Currently,the combination of transgenic technology and gene silencing technology has become a popular method to cultivate SMV resistant transgenic soybean.We have designed Micro RNAs(mi RNAs)against SMV.Mi RNAs and SMV were overexpressed simultaneously by agroinfiltration.The western blot results showed that when the mi RNA targeted at the 5' end of the positive-sense genemo of SMV,the accumulation of SMV CP was significantly inhibited.We also designed another mi RNA.The mi RNA could simultaneously target the 5' end of positive-sense genomes of SMV,Watermelon mosaic virus(WMV)and Bean common mosaic virus(BCMV).The anti-SMV effect of this mi RNA was identified in Nicotiana benthamiana.We cloned this mi RNA into soybean transgenic vector in order to obtain transgenic soybean resistant simultaneously to SMV,WMV and BCMV,which could contribute to the prevention and control of soybean mosaic virus disease.