Function Analysis Of Two Unknown Genes Of HaSNPV, Ha94 And Ha132

This paper reports the results of studies on sequence analysis of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) //mdlll-L fragment, function anaysis of HaSNPV two conserved genes, Ha94 and Hal32. This thesis encompasses four chapters.Chapter one is a general introduction of the research on baculovirus, including the classification status of baculovirus, characteristics of baculovirus genomes, functional reports of 29 completely conserved genes of baculovirus, application of baculovirus. The aim of this study is also included.The sequence analysis of the //mdlll-L fragment of the HaSNPV genome was reported in chapter two. The fragment contained five complete open reading frames (ORFs) representing ORF227 (a unique gene), a late expression factor 10 gene (/e/10), a baculovirus structural protein gene (vp1054), Ac55(a homologue of the AcMNPV ORF55) and Ac56 (a homologue of the AcMNPV ORF56), respectively. The detailed structural analysis of the 5 genes was reported.In chapter three, a conserved baculoviral gene of HaSNPV was identified. Sequence analysis indicated that Ha94 is 1086 bp long and encodes a putative protein of 362 amino acids with a predicted molecular size of 41.5 kDa. A late baculoviral transcription initiation motif ATAAG was found 65 nt upstream of the putative translational start site and a polyadenylation signal AATAAA was identified 14 nt downstream of the TAA stop codon. To elucidate its function, Ha94 was expressed as a GST-fusion protein in E. coli. The expressed protein was purified and used to generate antibodies in rabbits. The transcription and expression profiles of the putative gene were investigated in HzAMl cells. RT-PCR results suggested Ha94was a late gene. Western blot analysis of extracts of HaSNPV-infected HzAMl cells revealed a specific protein of 43 kDa from 48 h to 96 h p.i.. To investigate whether ORF94 is a structural component of HaSNPV, Western blot analysis of proteins in budded viruses (BVs) and occlusion derived virions (ODVs) was conducted. The protein was detected in ODV but not in BV, suggesting that orf94 encodes a structural component of ODVs. When ODVs were further fractionated into nucleocapsid and envelope components, the Western blot analysis indicated that the encoded protein was part of nucleocapsid and envelope. In summary, the data show that Ha94 encodes a novel ODV nucleocapsid and envelope protein (ODV-EC43) of HaSNPV.Chapter four is about the function elucidation of a conserved baculoviral gene Hal32. Sequence analysis indicated that Hal32 is 1152 bp long and encodes a putative protein of 384 amino acids with a predicted molecular size of 44.5 kDa. Computer-assisted analysis revealed there was a signal peptide near the N-terminal of this protein. Northern blot results suggested Hal32 is a late gene and produced multiple transcripts in different sizes. To elucidate its function, Hal32 was expressed as a GST-fusion protein in E. coli. The expressed protein was purified and used to generate antibodies in rabbits. Western blot analysis of extracts of HaSNPV-infected HzAMl cells revealed a specific protein of 43 kDa from 48 h to 96 h p.i.. To investigate whether HA 132 is a structural component of HaSNPV, Western blot analysis of proteins in budded viruses (BVs) and occlusion derived virions (ODVs) was conducted. The protein was not detected either in ODV or in BV, suggesting that HA132 is not a structural component of HaSNPV. A deletion recombinant virus (HaSNPVA132) of Hal32 was generated by homologous recombination in E. coli. Electron microscope pictures revealed the deletion virus could replicate in HzAMl cells, which indicates Hal32 is not essential for the replication of HaSNPV.