Inhibition Of BmNPV Replication In Bombyx Mori Cell BydsRNA Triggered RNA Interference

BmNPV often causes silkworm diseases and results in substantial economic losses for silkworm industry; thus it an important task to effectively prevent and cure silkworm diseases caused by BmNPV. RNA interference (RNAi) is a mechanism of gene regulation, which sequence-specifically degrades targeted mRNA. RNAi has been shown to protect against invading genetic elements such as transposons, transgenes and viruses, which potentially share a long dsRNA trigger. RNA-induced gene silencing offers a potentially useful method to inhibit viral replication. In theory, it can block viral replication effectively by synthetic dsRNAs or siRNAs, which target the indispensable genes of viral replication and thus silence the expression of target genes.To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435bp(Api), 300bp(Ap2) and 399bp(AM) in length against the various regions of BmNPV's DNA polymerase gene and DNA helicase gene, which are indispensable for viral replication in silkworm cells by TransMessenger?transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Api and AH can effectively suppress the replication of virus, but Apt had no effect on the inhibition of viral replication. Api and AH can reduce the infective liter of BmNPV with a peak change of approximately 3-4 logs on day 4 post-infection. DNA dot blotting showed that total DNA of normal silkworm cells as a positive contrast had no hybrid signal; however, total DNA of infected cells, which transfected Ap2 or AH had a very weak hybrid Signal by contrast with the negative contrast (dot a). The quantity of viral DNA in dot c and dot d reduced 72.3% and 63.4% compared with negative contrast by gel imaging system. The results of RT-PCR also indicated that when the dosage of dsRNA was 2 u g/well, the expression level of the two target genes (Ap2 and AH) were all obviously reduced 78.27% and 86.70% compared with the interior contrast while in the other dosages their expression level changed very little.In addition, the transfection efficiency and Cytotoxicity of TransMessenger Reagent and the distribution of introduced dsRNA in cells had also been studied in this thesis. Using Flow cytometer (FCM) we found that the transfection efficiency of TransMessenger Reagent was 60-70%. From the test of MTT, we also found that the volume of Bm-N shrunk, the cells' survival rate reduced, and there were many granules in cells when the dosage of transfection reagent exceeded 32 n L. FAM-labeled dsRNAs were transfected into silkworm cells and observed by fluorescence microscopy to determine their subcellular distribution pattern. We observed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 hours of transfection. Time course showed that the labeled dsRNAs were maintained in cultured silkworm cells for up to 5 days. After six days, the signal was too weak to be detected.In conclution, we can inhibite the replication of viral DNA effectively by synthetic dsRNAs or siRNAs, which target the indispensable genes of viral replication. The feasibility of dsRNA inhibiting BmNPV replication can not only give a model in the research of insect Nucleopolyhedroviru inhibition, but also gives some references onRNAi's application in the treatment of human viral diseases.