Resistance of transgenic silkworm to BmNPV could be improved by silencing ie-1 and lef-1 genes.

ABSTRACT

RNA interference (RNAi)-mediated viral inhibition has been used in several organisms for improving viral resistance. In the present study, we reported the use of transgenic RNAi in preventing Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in the transgenic silkworm B. mori. We targeted the BmNPV immediate-early-1 (ie-1) and late expression factor-1 (lef-1) genes in the transiently transfected BmN cells, in the stable transformed BmN cell line and in the transgenic silkworms. We generated four piggyBac-based vectors containing short double-stranded ie-1 RNA (sdsie-1), short double-stranded lef-1 RNA (sdslef-1), long double-stranded ie-1 RNA (ldsie-1) and both sdsie-1 and sdslef-1 (sds-ie1-lef1) expression cassettes. Strong viral repression was observed in the transiently transfected cells and in the stable transformed BmN cells transfected with sds-ie-1, sdslef-1, ldsie-1 or sds-ie-lef. The decrease of ie-1 mRNA level in the sds-ie1-lef1 transiently transfected cells was most obvious among the cells transfected with different vectors. The inhibitory effect of viral multiplication was decreased in a viral dose-dependent manner; the infection ratio of transfected cells for sds-ie-1, sdslef-1, ldsie-1 and sds-ie-lef decreased by 18.83%, 13.73%, 6.93% and 30.63%, respectively, compared with control cells 5 days after infection. We generated transgenic silkworms using transgenic vector piggyantiIE-lef1-neo with sds-ie1-lef1 expression cassette; the fourth instar larvae of transgenic silkworms of generation G5 exhibited stronger resistance to BmNPV, the mortalities for the transgenic silkworms and control silkworms were 60% and 100%, respectively, at 11 days after inoculation with BmNPV (10(6) occlusion bodies per ml). These results suggest that double-stranded RNA expression of essential genes of BmNPV is a feasible method for breeding silkworms with a high antiviral capacity.