Inhibition Of The Infection Of Rice Ragged Stunt Virus In Nilaparvata Lugens With RNA Interference Strategy

Rice ragged stunt virus (RRSV), a member of oryzavirus, is transmitted by brown planthopper in a persistent propagative manner, but not in transovarial manner. which caused the RRSV disease is severely affects rice production in south china. RNA interference is induces the degradation of target mRNA by the introduction of long dsRNA, which plays a fundamental role in antiviral defense in insect, including plant oryzavirus. The important gene which have the function of infection or replication with the RNAi mechanism to reduce it expression can offer a new method to control the virus disease. In this studies, we aim is find a important gene from RRSV and through the RNAi strategy to control the RRSV.In our studies, we choose the RRSV unstructure protein Pns6 and the structure protein P8 and P9, Under the T7 RiboMAXTM Express RNAi System condition in vitro synthesis the dsRNA. The dsRNA were mixed with 10% sugar applied to feeding Nilaparvata lugens as artificial diet, GFP dsRNA and the only 10% sugar as control. RT-qPCR method to detect the effection of dsRNA treatment after 6 d and 10 d, the results show that the Pns6 dsRNA have the best interference among the three genes. Next we use immunofluorescence microscopy technology observed the digestive system found that feeding Pns6 dsRNA have the best interference effectively also. Western blot method to detect the expression of RRSV encoding proteins after feeding the Pns6 dsRNA. The result showed that Pns6 dsRNA can interfere the proteins expression effectively, and we study the virus transmission after feeding the Pns6 dsRNA found that through the Pns6 dsRNA induce the RNAi mechanism can reduce the capacity of virus transmission.In conclusion, compared with the structure Protein P8 and P9 dsRNA from RRSV genes, the unstructure Protein Pns6 dsRNA have the best interference. In the early studies, we known that Pns6 is a multifunction protein, which including the movement protein, RNA silencing suppressor and Nucleic acid binding activity. In our lab, we found that Pns6 can colocalization with replication key protein Pns10, so we presumably the Pns6 also is the replication key protein. Choose the replication key proteins to induce the RNAi is a improtent way to control virus infected, Pns6 is the replication key protein that offer a important gene Pns6 come from RRSV and through the RNAi in order to control the RRSV spread with brown planthopper, which we can use the transplant expression the RRSV Pns6 dsRNA or through the Bacterial expression Pns6 dsRNA.