Study On The Fuction Of BmNPV IAP Protein

The iap(inhibitor of apotosis) is a widespread gene in all kinds of baculoviruses, which was first found during the research of screening p35 homologous genes, but with further studies, it was found that not all the baculovirual iap can inhibit cell apoptosis, it may have other multiple biological functions. Until now, two IAP proteins, IAP1 and IAP2, were found in Bombyx mori NPV genome, but its functions are still unknown.In this study, we took IAP1 and IAP2 as the research objects, iap1 and iap2 gene were deleted, respectively by Red recombination system to construct the gene deletion viruses: iap1-ko-bacmid and iap2-ko-bacmid; then iap1 and iap2 were repaired by Bac- to-Bac system to get the repaired viruses iap1-re-bacmid and iap2-ko-bacmid. The recombination bacmids were transfected into the Bm N cells, respectively. Identification of Bm N cell DNA fragmentation showed that IAP1 can not inhibit cell apoptosis induced by virus infection; real time q-PCR was used to analyze virtual genome replication level, transcriptional levels of early gene, late gene and very late gene of virus. The results showed that the deletion of iap1 had no remarkable effect on virus replication and transcription(P>0.05) while IAP2 played an important role in the inhibition of apoptosis(P<0.05) and it was essential for Bm NPV prolification.In order to further investigate the transcription mechanism of iap2, total RNA was extracted from silkworm fat body of fifth instar larvae infected by Bm NPV for five days, c DNA for silkworm gene expression library construction was generated by RT-PCR technique. 113 bp bait sequence of iap2 promoter was synthesized to generate the bait yeast strain which was used to screen the binding proteins by yeast one-hybrid system. Five DNA binding proteins were obtained, including LEF5, P6.9, BRO-D, ORF1629 and ODV-E25.In conclusion, our results showed that IAP1 had no remarkable effects on the virus transcription and replication. And it also had no effect on the inhibition of cell apoptosis. But IAP2 had the function of inhibition of cell apoptosis. Furthermore, we did a tentative exploration on the mechanism of transcription regulation of iap2. It will lay a foundation for further exploring the apoptosis resistance mechanism of IAP2 and provide new theory basis for the reformation of baculovirus vector construction..