Characterization Of ORF128 And ORF135 From Helicoverpa Armigera Single Nucleocapsid Nucleopolyhedrovirus (HaSNPV)

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is a single embedded NPV pathogenic to the bollworm, which is a major agricultural pest in many areas around the world and are attractive biological agent. The complete sequences of HaSNPV-G4 and Cl isolates have been reported. So far, the functions of several genes in HaSNPV had been characterized, such as polyhedrin, late gene expression factor 2 (lef-2), basic DNA-binding protein (BDBP), Hal22, Ha94, Ha29, Ha33 and Hal35, but the functions of many other genes remain unknown. In this paper, several genes were analyzed and the result prepared for the further functional study.Main results in this dissertation were as following 1. The preliminary analyses of the HaSNPV ORF128 geneIn HaSNPV-Cl genome HaI28 is located between 120,252 and 121,052 bp and encodes aputative protein of 266 amino acid residues with a predicted molecular weight of 30.5 kDa. Ha128homologues have been identified in all completely sequenced lepidopteran nucleopolyhedrovirus(NPV), but no homologue has been found in granulovirus (GV), and is thus considered as alepidopteran NPV-specific gene. Ha128 transcript in HzAM1 cells could be detected from 24 to 120h post-infection (p.i.) of HearSNPV by Northern blot. The Ha128 protein was detected at 24 h p.i.and remained detectable until 120 h p.i. by western blot using an anti-GST-Hal 28 antiserum. Theseresults suggested that Ha128 was a late gene in the infection cycle. The product of Hal28 was foundto be about 31 kDa, in agreement with the predicted molecular weight. Immunoflourescent assayshowed that Ha128 was located in cytoplasm. Protein pull-down and co-immunoprecitation analysisshowed that at least two potential host proteins interacted with Hal 28. In conclusion, Hal28 is a lateexpressed protein located in the cytoplasm and may interact with host proteins directly.2.The preliminary analyses of the AcMNPV ORF17 geneAutographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) is the type species of the Nucleopolyhedrovirus genus in the family Baculoviridae. The ORF17 ofAcMNPV(Acl7) is one of the core genes for all completely sequenced lepidopteran nucleopolyhedroviruses. In this study, we described this gene. A casein kinase II phosphorylation site (located at aa 69 - 72), two N-glycosylation sites (located at aa 78 - 81, 158 - 161) and a N-myristoylation site (located at aa 151 - 156) were found by Computer-assisted analysis. But no any significantly functional domain was found by protein searching tool PROSITE. RT-PCR analysis of Acl7 indicated that Acl7 transcript was firstly detected at 3 h.p.i. and remained detectable until 72 h.p.i., which is in agreement with the early promoter motifs. The result suggested that Acl7 might be an early gene during virus infection cycle. The product of Ac 17 was analyzed by Western blot using anti-Acl7 antiserum and the result revealed that the Acl7 protein was detected at 6 h.p.i and remained detectable onwards. The product of Ac17 was about 19kDa in size, consistent with predicted 18.5 kDa molecular weight, suggesting that no major post translation modification of the Ac17 protein occurred in host cells.3.The preliminary analyses of the HaSNPV ORF135 geneThe ORF135 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV)(Ha135) is one of the 20 genes that are unique to HaSNPV. Computer-assisted analysis revealed that four potential post translation modification sites, four transcription factor associated domains and a DNA binding protein domain were found in Hal35 amino acid sequence. Northern blot analysis of Hal35 indicated that Hal35 transcript was detected at 12 h.p.i.and remained detectable at up to 122 h.p.i. RT-PCR method was used to understand the temporal regulation of the transcript at earlier stages, the result showed that the Ha135 transcript was detected as early as 3 h p.i., suggesting that Ha135 was an early gene, which is in agreement with the early promoter motifs. The Hal35 protein was also detected at 12 h.p.i and remained detectable until 122 h.p.i. by western blot using an anti- Ha135 antiserum. The product of Ha135 was found to be about 29 kDa, bigger than the predicted 24 kDa molecular weight, suggesting that post translational modification of the Ha135 protein occur in host cells. The subcellular location was studied using EGFP-Ha135, which suggested that the Ha135 protein is primarily localized in the nucleus, which is compatible with several functional domains present in Ha135 amino acid sequence. Together, these results suggest the possibility that HearSNPV ORF135 might be involved in viral DNA transcription and /or replication.4.The expression and subcellular location of Ha99, Ha101, Ha102 and p6.9 gene in Tn cellsHa99, Ha102, Ha101 and p6.9 genes were inserted into pFastBacHTe to constructed pFastBacHTeHa99, pFastBacHTeHa102, pFastBacHTeHa101 and pFastBacHTep6.9, respectively. The recombinant donor plasmids were transformed into DH10 competent cells and constructed recombinant Bacmids. Four recombinant Bacmids were transfected into Tn-5B1-4 cells of the cabbage looper, Trichopolusia ni. SDS-PAGE analysis showed the expression products of Ha99, Ha102, HalOl and p6.9 were 44, 40, 60 and 40kD, respectively, consistent with their predicted molecular might, suggesting that no major post-translation modifications occurred in these proteins. The subcellular locations were studied by EGFP fusion protein expressed in Tn-5B1-4 cells. The results indicated that the Ha99-EGFP and Ha102-EGFP fusion protein located in the cytoplasm of host cells, while the HalOl-EGFP and p6.9-GFP fusion protein located in the nucleolus of host cells. The transcript analyses were performed by RT-PCR, the results suggested that the Ha99 transcript was detected in 6 h p.i. and remained detectable until 122 hp.i and Ha102 transcript was first detected in 3 h p.i. and was detectable until 122 hp.i, suggesting that both genes might be earlier genes in the infection cycles, consistent with the early promoter motifs in their nucleic acid sequences. HalOl transcript was first detected in 24 h p.i. and remained detectable until 122 hp.i, suggesting that HalOl gene might be a late gene in the infection cycles, consistent with the late promoter motif in their nucleic acid sequences.