Development And Application Of Anti-TMV RNAi Nanomeds Based On DsRNA-nano

Double-stranded RNA can interfere the gene expression of tobacco mosaic virus,but dsRNA can be degraded easily by nuclease,and the stability can be improved by coating it with nano-material HACC,in view of the lack of studies on the absorption process of nano-material in plants,this study focused on comparing the effects of three drug application methods and transport routes.The main findings are as follows:（1）TMV-encoded CP,MP and Rd RP genes were used as the target sequences to transcribe and synthesize corresponding dsRNA in vitro,the expression of TMV-30B in Nicotiana benthamiana and the hypersensitive necrosis response in Nicotiana tabacum var.Samsun NN were studied using CP gene,RNA and protein levels as markers,by comparing the inhibitory effect of six target sequences corresponding to dsRNA on TMV,Rd RP1461-1774 was screened.（2）The prokaryotic expression vector of TMV Rd RP1461-1774 gene was constructed according to the high efficient sequence.The dsRNA was induced and expressed by HT115 strain.The optimal culture condition was 37℃until OD₆₀₀value was 0.4,after 5 hours of IPTG induction with the final concentration of 0.4 mmol/l,dsRNA was soaked into Nicotiana benthamiana leaves for Small RNA sequencing.The results of Small RNA sequencing showed that in the RNAi process induced by PBS immersion,the sense chain and the antisense chain produced si RNA at approximately equal frequency in the control group,while the exogenous dsRNA immersed in the exposed group resulted in the enrichment of si RNA in the target region,which indicated that the application of exogenous dsRNA could cause the change of si RNA expression abundance.（3）The HACC-dsRNA nanoparticle complexes was synthesized by fusing the dsRNA expressed in prokaryotic cell with the nano-material of quatemized chitosan,The structur used transmission electron microscopy and atomic force microscopy to study the structure and properties of nanoparticles.and it was found that the nanoparticles were uniform in size in the range of 30-50 nm,and showed spherical or oblate shape.The maximum loading rate of dsRNA and HACC was 1:0.8,With the increase of storage time and temperature,the release efficiency of HACC-dsRNA increased.（4）Small RNA from tobacco treated with nanoparticle-dsRNA complexes by injection,spraying and root irrigation,and found that the amount of Small RNA produced by injection and spraying was much higher than that by root irrigation.Combined with q RT-PCR,the relative expression level of 4dpi treated group was lower than that of Control Group,and the relative expression level of 4dpi treated group was also lower than that of control group,in the treatment group,the virus expression in the top leaf was relatively low,so it was considered that the virus blocking effect was the best in the treatment group.（5）Ds RNA was labeled by Cy3-d CTP,HACC was labeled by FITC,and the transport route of nanoparticle-dsRNA complexes was observed by confocal laser scanning,it includes the process of nanoparticle application from root and uptake by cells,and the preparation of protoplast to verify the absorption of nanoparticle-dsRNA complexes.Studies have shown that the cells absorb nanoparticles through adsorption and phagocytosis and direct permeation of nanoparticles,and the injection and spraying treatment group has stronger fluorescence,in the root irrigation treatment group,the nanoparticles accumulated in the leaves with time,The process of drug absorption and conduction through xylem was observed through the Cross section and Longitudinal section of root and stem.The results showed that Chitosan quaternary ammonium salt had a good effect on dsRNA slow release,protect and improve the stability of nucleic acid.The research results are of great significance in controlling plant virus diseases by RNA interference.