The Effect On BmNPV Proliferation In Vitro By Transgenic Interfere Gp64 And Lef-1 Gene Fragments

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most common and damaging pathogeny in the silkworm, which does great harm to sericulture once explosion. So the research on how to enhance the resistance of silkworm to BmNPV is of great scientific and practical value. With the completion of whole genome sequence of BmNPV, we know the pathogeny more clearly that some essential genes are criticality for BmNPV propagation. Therefore, inhibition of the essential genes functional expression is an effective method to prevent silkworm from suffering BmNPV. RNA interference (RNAi) is a highly conserved mechanism of virus resistence in which double-stranded RNA (dsRNA) mediates specific degradation of the target mRNA. We choose two genes gp64 and lef-1 which are essential for baculovirus propagation as suppressing target genes, investigating the inhibition effects of the corresponding dsRNAs on the replication and propagation of BmNPV in vitro at the cell level by the combination of RNAi and transgenic technology. It would be a brand-new method for breeding antiviral silkworm strain in the future. The major findings are as follows:.1. Cloning of target genes and construction of transgenic RNAi vectorsBased on the genome sequence of BmNPV, we cloned fragments of+233～+867 region of gp64 ORF and +85～+754 region of lef-1 ORF respectively.Then we cloned the tested fragments into frame vetors contain two different promoters 39KP and LEF3P to construct four piggyBac-based transgenic RNAi vectors and named them as pigA3-EGFP-39KPgp64, pigA3-EGFP-39KPlef1, pigA3-EGFP-LEF3Pgp64 and pigA3-EGFP-LEF3Plef1 respectively.2. Establishment of transgenic cell lines which express corresponding dsRNA of target genes Two different cell lines BmE and BmN-SWU1 were transfected with the constructed transgenic RNAi vectors. The transfected cells were elected under G418 pressure for about three months and fluorescence cell ratio raised from about 10% up to 70% and 80% eventually.3. The effect on BmNPV proliferation in vitro with the corresponding dsRNA of target genesThe dsRNA of target genes has significant inhibition effects on the replication and propagation of BmNPV. The effect of corresponding dsRNA of gp64 is better than that of lef-1 gene. Under the virus concentration of 10-3 and 72h after infection,the Bm-BacPAK6 virus propagation in transfected cells were about 50% lower than that in normal cells. The propagation of wild-type BmNPV was also significantly inhibited in transgenic cells:The infection rate and the number of generated polyhedra in fluorescence cells are both lower than in normal cells which do not fluoresce.