Zhang Yaozhou

Inhibition of BmNPV replication in Bombyx mori cell by dsRNA triggered RNA interference

RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435 bp(Ap1), 300 bp(Ap2) and 399 bp(AH) 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 TransMessengerTM transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Ap2 and AH can effectively suppress the replication of virus, but Ap1 had no effect on the inhibition of viral replication. Ap2 and AH can reduce the infective titer of BmNPV with a peak change of approximately 3–4 logs on day 4 post-infection. The results of reverse transcript polymerase chain reaction (RT-PCR) and DNA dot blotting also indicated that the expression level of the two target genes and the quantity of viral DNA both distinctly decreased under the influence of Ap2 or AH. Furthermore, using fluorescence microscopy we analyzed the distribution patterns of dsRNA. Our studies revealed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 h of transfection.