| Silkworm is an important economic insect. BmNPV (Bombyx mori L nucleopolyhedrovirus) is a key pathogeny to silkworm with highly infective. And the reciprocity between virus and host is an important issue which was paid many attentions by researchers all the time. Mechanism associated with BmNPV resistance has not yet entirely clear, but it could be attributed to both host and viral.In this study, a conserved gene (Bm5) in Lepidoptera baculoviruses was characterized from the transcription,expression,cellular location and knockout. The object of this study is to enhance our understanding of baculovirus molecular biology. We also screened genes associated with BmNPV resistance by using the Bombyx mori high-density Oligo microarray. The Microarray provided the whole genomic information of Bombyx mori, breaking the research limitations of single gene. In addition, we evaluated the datas by GO and Pathway, and further identified differentially expressed genes by using semi-quantitative PCR and fluorescence quantitative PCR. We hoped to elucidate the resistance mechanism. The results are as following:1. Orf5(Bm5) was located at nt 4,605-5,600 of BmNPV genome, containing 993 bp and coding 331 aa with a predicted molecular weight of 39.3 kDa. Bm5 was the homology sequence of AcMNPV orf13. The late transcription motif of baculoviridae, ATAAG was located at the upstream of ATG. And the transcription terminal signal, AATAAA was found at the downstream of the terminal codon TAA. Two primers were designed as the Bm5 gene sequence and used for PCR amplification. PCR products were cloned into pET30a(+) vector and Bm5 were expressed as fusion protein. The purified fusion proteins were injected into New Zealand white rabbits to harvest polyclonal antibodies. Western blot analysis of extracts from BmNPV-infected BmN cells revealed a specific polypeptide with an apparent size of 39.3 kDa when using the BM5 antiserum.2. The RT-PCR resulted showed that Bm5 gene was transcribed in the infected cells from 12 to 72 h. Bm5 fusion protein was expressed using the E. coli expression system and polyclonal antibodies were harvested. The western blot analysis showed that Bm5 was detected in the infected cells from 24 to 72 h, which further suggesting that Bm5 is a late gene. The detected BM5 had a molecular weight of 39.3 kDa, same as the predicted one, which suggesting that BM5 have no post-transcription modification. BM5 was not detected either in budded viruses or occlusion-derived virus. The sub-cellular localization of the BM5 proteins was investigated by immunofluorescence. The fluorescence was concentrated on the cytoplasm and no fluorescence was detected in the nucleoplasm.3. Bm5 was rapidly disrupted in E.cori by Red recombinant system. BmNPV which disrupted Bm5 was rescued by the BmNPV/Bac to Bac system and Red system. vBm5-Wt,vBm5-Ko and vBm5-Re viruses were constructed respectively. The results indicated the BmNPV Bac-to-Bac expression system can effectively express foreign gene.4. We screened 93 genes (44 up-regulation genes,49 down-regulation genes) associated with BmNPV resistance by using the Bombyx mori high-density Oligo microarray. The results of microarray were analyzed by Gene Ontology (2.0 version), which showed that 51 gene (24 up-regulation genes,27 down-regulation genes) have functional annotations. Molecular function of GO included enzyme activity,oxidoreductase activity,binding,structural constituen,transporter activity. Biological process included metabolism proteolysis,electron transport,protein targeting,transport,protein biosynthesis. Cellular components included intracellular,membrane,ribosome,extracellular region. Results of Pathway analysis included metabolism,fructose and mannose metabolism,acid metabolism,ribosome,oxidative phosphorylation,pentose phosphate pathway,glycolysis/gluconeogenesis,citric acid cycle (TCA cycle) and so on.5. We identified 10 differentially expressed genes (7 up-regulation,3 down-regulation) by Semi-quantitative PCR and fluorescence quantitative PCR. Results Showed the expression levels in 5 of 7 up-regulation genes were higher in NB and BC8 than in 306, which identified as up-regulation genes associated with BmNPV resistance,2 of 7 up-regulation genes were higher in BC8 than in NB. The expression levels in all 3 down-regulation genes were higher in 306 than in NB and BC8, which identified as down-regulation genes associated with BmNPV resistance. The results further confirmed the reliability of the chip...
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