Study On Lily Mottle Virus DsRNA-mediated Disease Resistance

The antiviral strategy of prokaryotic expression of viral pathogens dsRNA is safer and more convenient than plant antiviral genetic engineering,and saves time and effort compared with in vitro synthesis of dsRNA.Lily is severely infected with Lily mottle virus(LMoV).The current prevention and control measures are mainly traditional detoxification and chemical treatment,and lily as monocotyledon is difficult to get resistance plant through transgenic technology.In this study,dsRNA derived from LMoV coat protein(CP)and columnar inclusion protein(CI)gene sequences were obtained for the first time by prokaryotic expression system,and we demonstrated the feasibility of this method in against LMo V.The positive and negative fragments of the target gene were inserted into on both sides of the Pdk intron fragment of plant expression vector pRNAi-LIC by One-step,zero-background ligation-independent cloning(OZ-LIC)method and three RNAi vectors were successfully constructed respectively.Antiviral analysis was performed by Agrobacterium-mediated transient-expression assay.The results of RT-PCR and indirect ELISA suggest that the resistance to LMoV conferred by the CI517 and CI295 hpRNAs were better than CP400 hpRNA,indicating that the ihpRNA structure can trigger RNAi.Recombinant piasmids were constructed by ligating the three hairpin constructs with the pSP73 prokaryotic expression vector and were transformed individually into the E.coli M-Jm109 lacY,RNase III-deficient strain,and dsRNA was induced by IPTG.The crude extracts of dsRNA were obtained by NANO HOMOGENIZE MACHINE,and were applied to lily plants as a spray and the experiment confirmed a preventative efficacy.The results of Real-time PCR and RT-PCR demonstrated that CI517-dsRNA and CI295-dsRNA could protect lily from disease or delay disease progress.There was no diseased strain in the dsRNA treatment group throughout the growth period,and the amount of LMoV accumulation in the lily of symptom was lower than that in the control group,indicating that ds RNA derived from LMoV had a very good protective effect on lily.