Characterization Of Bm61 And Orf74 From Bombyx Mori Nucleopolyhedrovirus

The family Baculoviridae is a highiy selective pathogen in arhropods, mainly in insects of the order Lepidoptera.So far,the genomes of 47 baculoviruses have been sequeneced.And it is revealed that 30 genes are baculovirus core genes and 62 genes are conserved in Lepidoptera baculoviruses after analyzing the whole genomes.Bombyx mori nueleopolyhedrovirus is one of most studied baeuloviruses;some genes have not been characterized.In this study,a non-conserved genes(orf74) and a conserved genes(Bm61) in Lepidoptera baculoviruses are characterized.These two genes were characterized from the transcription,expression,cellular location and knockout.The object of this study is to enhance our understanding of baculovirus molecular biology.The results are as following:1.orf61(Bm61) is located at nt 61,188—61,892 of BmNPV genome, containing 399 bp and coding 133 aa with a predicted molecular weight of 15.5 kDa.Bm61 is the homology sequence of AcMNPV的orf75.The late transcription motif of baculoviridae,ATAAG was located at the upstream of ATG.And the transcription terminal signal,AATAAA was found at the downstream of the terminal codon TAA.2.The RT-PCR results showed that Bm61 genes was transcribed in the infected cells from 12h to 72h.Bm61 fusion protein was expressed using the E.coli expression system and polyclonal antibodies were harvested. The western blot analysis showed that Bm61 was detected in the infected cells from 12 to 72 h,which further suggesting that Bm61 is a late gene. The detected Bm61 had a molecular weight of 15.5 kDa,same as the predicted one,which suggesting that Bm61 have no post-transcription modification.The sub-cellular localization of the Bm61 proteins was investigated by immunofluorescence.It was found that the proteins were located on the nuclear membrane and the nuclear ring zone. 3.Bm61 deletion Bacmid was generated by Red-recombination in E. coli DH10B.A fragment of Bm61 gene was knocked out from the BmNPV Bacmid and the Cm gene was inserted into the same site.The gfp and polh genes were transposed into the recombinant Bacmid which was used to transfect BmN cells.Cells transfected with Bm61-knockout Bacmid showed a few fluorescent spots,which indicated that there was no spread of the virus beyond the cells transfected with Bm61 deletion Bacmid.The results of PCR showed that there was no BV in the supernatant of transfected cells,which indicated that it reduced the production of BV induced of the deletion of Bm61.However,Bm61-recovered Bacmid showed the same feature of wild type Bacmid.The deletion of Bm61 did not affect the replication of the virus genome and the transcription of gp64.Therefore,it was concluded that the Bm61 gene was an essential gene for BV production.4.orf74 is located at nt 69,987-70,449 of BmNPV genome(nt), contains 462 bp and codes 154 aa,with a predicted molecular weight of 17.3 kDa.The late transcription motif of baculoviridae,TTAAG was found at 9 nts.orf74 is the homology sequence of AcMNPVorf91.5.The results of RT-PCR showed thal orf74 genes was transcribed in the infected cells from 12h to 96h,which indicated that orf74 was probably a late gene.ORF74 fused with GFP was used to investigate its subcellular location.ORF74 was found to be located in the nucleus.6.orf74 deletion Bacmid,vBm-ko was generated by Red-recombination in E.coli DH10B.The analysis of orf74-knockout virus replication in BmN cells showed that the deletion of orf74 did not affect both the reproduction and the replication of the virus genome in BmN cells. The bioassay indicated that the deletion of off74 could extend the survival time of the infected larvae,but the yield of virus had no changes.These results showed that orf74 was a non-essential gene for virus replication,but related to the virulence.