Research On Development Of Tobacco Mosaic Virus RNAi Preparation

Tobacco mosaic virus(TMV)disease is an important disease in vegetables and other crops.At present,there is a lack of effective control measures,RNA interference(RNAi)technology to provide a new idea for the prevention and treatment of virus diseases.Studies have shown that exogenous double-stranded RNAs based on virus genes can induce RNAi in plants,but dsRNA is easy to be degraded and limited in time,which limits the application of this technology.In this paper,the prokaryotic expression vector of TMV CP gene was constructed by using the TMV coat protein protein(TMV)gene as the target fragment.DsRNAs were induced by(E.coli)HT115(RNase III)and dsRNA was synthesized in vitro.The synthesized dsRNA was fused with two kinds of nanomaterials,such as chitosan(CS)and layered double hydroxide(LDH)to form preparation CS-dsRNA and LDH-dsRNA.The results of stability test showed that the amount of dsRNA decreased by 80.01%,58.89% and 59.76% respectively at 4 ℃,25 ℃ and 37 ℃ for 30 days.The degradation rate of dsRNA at 4 ℃ was slightly higher than that at 25 ℃ and 37 ℃.The amount of dsRNA in CS-dsRNA preparation increased by 65.98%,103.82% and 157%,respectively,and the amount of dsRNA in LDH-dsRNA preparation increased by 54.30%,58.4% and 66.42%,respectively.And with the increase of temperature,the amount of dsRNA increased,indicating that the nanocomposite dsRNA has a slow release effect.Our results show that the nano-materials(CS and LDH)play a good role in the protection,slow release and stability of dsRNA.Using greenhouse potted of half leaf method experiment result showed that the preventive effects of induced dsRNA expression,dsRNA synthesized in vitro,CS-dsRNA and LDH-dsRNA on TMV were 45.63%,60.12%,57.79% and 55.18%,and the therapeutic effects were 48.26%,65.01%,58.4% and 59.08%,respectively.The results were of great significance to the prevention and treatment of plant virus diseases by using RNA interference.