Functional Analysis Of Vp80, Bm21 Of BmNPV And Sumo Of Bombyx Mori

The BMNPV genome was 128413 bp nucleotide long and contained 136 openreading frames (ORFs). In this study, Bmvp80 which is located at both BV and ODVof Bombyx mori nucleopolyhedrovirus (BmNPV), BmNPV orf21 (Bm21) which is theunique gene of group I NPVs, were characterized. At the same time, the characters ofSUMO gene of silkworm and the effect of over-expression of BmSUMO on BmNPVreplication in BmN cells were analyzed. The results were as following:1. study of Bmvp80Partial coding region of the Bmvp80 was cloned and expressed in E.coli BL21, andantiserum against BmVP80 was prepared. The sub-cellular localization of theBmVP80 proteins was investigated by immmunofluorescence. The fluorescence wasconcentrated within the nucleoplasm. A BmNPV Bacmid with the Bmvp80 genedisrupted was constructed using the ET-recombination system to investigate the roleof Bmvp80 during the baculovirus life cycle. Disruption of Bmvp80 resulted in singlecell infection phenotype, whereas a rescue BmBacmid restored BV titers to wild typelevels; however, the homologous gene Acvp80 from AcMNPV could not complementthe BmBacmid lacking a functional Bmvp80 gene. Electron microscopy of cellsinfected with BmNPV lacking functional Bmvp80 revealed that the number ofnucleocapsids was markedly lower. These results suggest that Bmvp80 is essential fornormal BV production and nucleocapsid maturation, and is functionally divergentbetween baculovirus species.2. analysis of Bm21The open reading frame 21 (Bm21) of BmNPV was one of the unique genes ofgroup I NPVs. In this study Bm21 was cloned and expressed. The RT-PCR analysis ofBm21 demonstrated that Bm21 transcribed as early as 12h p.i. and kept highly steadyat late phase. The Bm21 was located at both the nucleoplasm and the cytoplasm. ABmNPV Bacmid with the Bm21 gene disrupted was constructed using theET-recombination system, no obvious cytological change was observed in transfectedcells, which means the knockout of Bm21 may influence the replication of BmNPV.3. analysis of BmSUMO The SUMO of Bombyx mori (BmSUMO) contains 91 amino acids and the codingregion was interrupted by two introns. The BmSUMO was more similar to vertebrateSUMO-2/3, sharing 67% sequence identity with human sumo-2/3, while share only51.6% identity with human sumo-1. In BmN cells, many proteins were modified byBmSUMO peptide. Immunofluorescence microscopy demonstrated that theBmSUMO was present within the whole BmN cells and also aggregated as dots in thenucleus. The over-expression of BmSUMO under ie-1 promoter inhibited the growthof Bombyx mori nucleopolyhedrovirus (BmNPV) before the 48 p.i. of virusreplication cycle.