Dissertation > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens

Functional Analysis of BmNPV ORF9 and ORF56

Author YangZhangNv
Tutor ZhangChuanXi
School Zhejiang University
Course Agricultural Entomology and Pest Control
Keywords Bombyx mori nucleopolyhedrovirus baculovirus Bm9 Bm56 gene function gene knock out microarray
CLC S476.1
Type PhD thesis
Year 2008
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Baculoviruses are extensively being studied for potential use in bioinsecticides around the world and as expression vectors for heterologous gene expression in insect-derived cells as well as in the host caterpillars. Recently, Bombyx mori nucleopolyherovirus (BmNPV), has been successfully developed for surface display. Additionally, baculoviruses are regarded as potentially useful gene therapy vectors. These applications drive the study of molecular basis of baculovirus infection.Since the complete genome of BmNPV was determined, extensive studies have focused on the function of individual gene .BmNPV ORF9 (Bm9), homologous to ORF17 of AcMNPV, is considered as a gene specific for Lepidopteran NPVs, suggesting that the gene plays an important role in the infection cycle of lepidopteran baculovirus. In the present study, the role of the Bm9 gene product was investigated in BmNPV-infected cells. The BmNPV bacmid was utilized to generate a Bm9 knockout virus in E. coli. There was a significant reduction of infectious BV production in cells infected with the Bm9 null virus relative to a wt (wild type) virus, however, the kinetics of viral DNA replication was unaffected. Subsequently, the defect of BV production was recovered by the Bm9 rescue bacmid by reinserting the Bm9 gene cassette into the polyhedrin locus of the same virus genome. And the polyhedra formation was not affected by the deletion of Bm9 via Electron microscopic observation. The B. mori larvae bioassay showed that the BV for Bm9 null virus had an extended insect-killing time and an increased LD50, compared to wt virus, indicating that the Bm9 is an important factor related to virus infectivity. Thus, the above data showed that the bm9 plays an important role in virus infection both in vitro and in vivo, though it is not essential for virus replication.BmNPV ORF56 (Bm56) is considered to be a core gene, with homologues existing in all baculoviruses that have had their genomes sequenced to date,including lepidopteran NPVs, lepidopteran granuloviruses,hymenopteran NPVs and a dipteran baculovirus. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopyrevealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells, however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50% lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo, however, it is not essential for BV production in vitro.We constructed an oligonucleotide microarray consisting of 60mer oligonucleotides, which represents 136 described genes of the BmNPV genome. Then the microarray was used to analysis the temporal transcription of BmNPV genome. Additionally, we used oligonucleotide microarray representing the BmNPV genome combined with quantitative PCR analysis to investigate global effects of Bm9 deletion on viral gene transcription at 6 h p.i.. We detected lef11, egt, ORF35, ubquitin, bro-b and dbp dramatically reduced in mRNA level from cells infected with BmBacKO-PG. ORF45 and ORF44 showed modest decrease, with statistical significance, from BmBacKO-PG. Simultaneously, p6.9, bro-d, ORF67, helicase, pe38, ie-2 and ie-1 showed modest increase in the absence of Bm9. This result lays a basis for the further research on the baculovirus functional genomics.

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