Research Of DsRNA Expressed By Bacteria To Protect Rice From RBSDV And RSV

Rice is an important feed crop in our daily lives. So cultivating healthy rice played an important role in the filed of ensuring the safety of food security production. Rice Black-streaked Dwarf and Rice Stripe Disease which were infected by RBSDV and RSV respectively. The two diseases cause a serious loss to rice production in our country. Chemical control still was primary control strategy of rice viral diseases in our country, but it had many problems, such as pesticide residue, environmental pollution and accumulating resistance. So exploring a new antivirus system was of utmost urgency. Prokaryotic expression of dsRNA can induce potent and specific gene silencing, which called RNA interference (RNAi). RNAi is a plant self-resistant reaction and a process of sequence-specific gene silencing in animals and plants. PTGS plays an important role in defense mechanism of plants, and dsRNA homologous to the silenced genes. So two hot-spot gene sequences from RBSDV (KF576666) and RSV (FM958476) were selected and two hpRNA vectors were constructed. This work explores the optimum induction conditions for high-efficiency output dsRNA and the most effective way of delivering to Oryza sativa. This part experiment was conducted in various combination conditions of temperatures, IPTG concentration and inducing times. Then dsRNA was extracted by the way of Trizol method. Concentration of inducing dsRNA (at OD260) was measured by ultraviolet spectrophotometer. Concentration of dsRNA=dilution ratio×A260×0.04. The result showed that dsRNA with the condition,32℃、3h、0.4mmol/L IPTG can gain high output. The result of experiment in rice field showed. The conderation of pGEM-RSVCP400expression vector was2.4×10-3μg/μL which can against RBSDV infection in the best level, with the lowest morbidity being25.1%. The conderation of pGEM-RSVCP400expression vector was2.4×10-3μg/μL which can against RSV infection in the best level, with the lowest morbidity being11.7%. Northern blot showed that the resistance was an RNA-mediated virus resistance. Our findings indicate that dsRNA could be an effective tool that protects Oryza sativa from RBSDV and RSV infections.