Orf60 Gene Has Regulation Effecs On Bombyx Mori Nucleopolyhedrovirus Replication And Transcription

As is known to all,The polyhedrin gene promoter of Bombyx mori nucleopolyhedrovirus(BmNPV)offer a super-efficient start function.it is often used as the insect baculovirus expression system launching the exogenous gene expression.We have screened a variety of protein factors that can interact with the polyhedrin gene promoter by Yeast one-hybrid system.Among them the ORF60 is regarded as one kind of DNA binding protein of BmNPV,which may have the function of regulating virus gene transcription,but its detailed functions still remain unknown.Red recombination technique is a common method that used to study baculovirus gene function by homologous recombination and Bac-to-Bac system can repair the purpose gene by Fixed-point translocation.In this study,we generated orf60-ko-Bacmid and orf60-re-Bacmid by the above two methods,respectively.Then the same amount of DNA extract from three kinds of Bacmids were transfected into normal BmN cells,respectively.The results of viral titer and Real-Time PCR revealed that orf60 gene is the essential gene for viral genome replication and it would impact on the assemble of virus particles.Then the transcriptional phases of orf60 gene in Bm N cells which infected together with BmNPV were analyzed by qPCR.The results revealed that the transcript of orf60 gene was first detected at 12 h post-infection.Comparing with that of three kinds of typical genes about lef3,vp39 and p10,we come to the conclution that orf60 is one kind of late gene of BmNPV.Furthermore,The transcriptional level of three kinds of typical genes form orf60-ko-Bacmid all less than below orf60-re-Bacmid and wtBacmid significantly(p<0.01).In order to explore how the orf60 regulate polyhedrin gene promoter,we compare the quantitative detection of luciferase activity.The results show that the delection of orf60 gene would result in reducing the activity of polyhedrin promoter.