Construction Of Reverse Genetics System Of Bm BDV, Bm CPV And Study On Two Silkworm Strains With Different Resistance To Bm CPV’s Infection

Bombyx mori bidensovirus(Bm BDV) can specially infect columnar cell of silkworm’s midgut, and causes silkworm flecherie. The genome of Bm BDV China Zhenjiang isolate(Bm BDV-Z) consists of two non-homologous single-stranded linear DNA molecules VD1(6,543 nts) and VD2(6,022 nts), which are encapsidated into separate virion. At present, there is no cell line in vitro for Bm BDV infection, thus we cannot study on its gene function through genetic manipulation. The genome of Bm CPV is composed of 10 ds RNA segments and each segment has a complete open reading frame(ORF). At present, we are unable to study on Bm CPV’s gene function by mehod of gene knock-out or gene knock-in due to a lack of sensitive cell line, as a result, study on gene function of Bm CPV had little progress these years. Various silkworm strain has different resistance to Bm CPV, but the resistance difference between different silkworm strain is unclear. In view of the status quo and existing problems, we carried out study on rescue of Bm BDV based on Bm BDV genome DNA, on rescue of Bm CPV based on using Bm CPV RNA to transfect Bm N cell, and expected to support new platforms for studying on gene function of Bm BDV and Bm CPV.1、Study on reverse genetics system of Bm BDVTo build a platform for studying on gene function of Bm BDV, recombinant plasmids p GEM-VD1 containing VD1 genome were transfected into Bm N cells of silkworm, Bombyx mori, the transfected cells displayed obvious pathological changes, the structural proteins of Bm BDV can be detected with Western blotting and immunofluorescence assay, indicating p GEM-VD1 replicated in the trnsfected Bm N cells and viral proteins of Bm BDV were expressed. Furthermore, a recombinant baculovirus Bm Bac-VD1 inserted with Bm BDV VD1 genome was constructed, the Bm N cells showed apparent pathological changes after being infected with Bm Bac-VD1 genome. Results of both Western blotting and immunofluorescence assay indicated the viral structural proteins of Bm BDV were expressed in the Bm Bac-VD1-infected Bm N cells, meanwhile baculiform and spherical virion were observed in infected cells by electron microscopy, moreover, the two kinds of virion were separated by differential centrifugation, results of molecular biological detection revealed Bm Bac-VD1 DNA was packaged within spherical virion. Results above suggested that Bm BDV can be rescued based on plasmid inserted with Bm BDV genome, Bm BDV-like virus particles could be obtained by baculovirus containing Bm BDV VD1 genome, indicated that we have constructed a reverse genetics system with which gene function can be studied through gene deletion or gene mutation.2、Study on reverse genetics system of Bm CPVWe used segment assembly and homologous recombination to separately clone each whole length c DNA of Bm CPV 10 ds RNA into p IZT-V5/His vector and obtained recombinant plasmids p IZT-CS1—p IZT-CS10. Meanwhile, Bm CPV S10 segment’s ORF encoding polyhedron was replaced by Ds Red gene to construct recombinant plasmid p IZT-CS10(Ds Red).In this study, we transfected linearized recombinant plasmids containing c DNA of Bm CPV genome segment into Bm N cell, and apparent pathological change was detected. Bm CPV expressed proteins were identified by Western blotting and immunofluorescence, real time PCR result displayed that Bm CPV’s gene up-regulated. Furthermore, Bm CPV S1-S9 m RNA and S10(Ds Red) m RNA were transfected into Bm N cell,and the supernatant of the transfected cells were transfered to inoculate normal cell 120 h post transfection, red fluorescence could be detected and the ratio of cell showing red fluorescence increased over time, indicating S10 segment is essential for Bm CPV replication, the site with which virus capsid protein recognized and embed S10 segment is within the non-coding region. Results above suggested that the system we constructed by transfecting Bm CPV genome RNA from in vitro transcription into Bm N cell can be used to study on Bm CPV’s gene function.Based on this system, recombinant Bm CPV can be constructed through inserting foreign gene into gene segment nonessential for virus replication, and new-type recombinant CPV insecticide can be developed by similar strategy.3、Study on comparative transcriptome of midguts of different silkworm strains infected with Bm CPVHigh-throughput paired-end RNA sequencing(RNA-Seq) was performed to investigate the gene expression profile of a resistant Bombyx mori strain, Ou17, and a susceptible strain, Lan5, which were both orally infected with Bombyx mori cypovirus(Bm CPV) in the midgut. There were 330 and 218 up-regulated genes, while 147 and 260 down-regulated genes in the Lan5 and Ou17 strains, respectively.Ontology(GO) enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis for differentially expressed genes(DEGs) were carried out. Moreover, gene interaction networks(STRING) analyses were performed to analyze the relationships among the shared DEGs. Some of these genes were related and formed a large network, in which cuticular proteins(Cpr62Bc) and minichromosome maintenance 5 protein(DNA replication licensing factor Mcm5) were key genes among the common up-regulated DEGs, whereas heat shock protein 23(Hsp23) was the central gene among the shared down-regulated DEGs between Lan5 vs Lan5-CPV and Ou17 vs Ou17-CPV.Our study offers new clews for further exploring the immune mechanism of silkworm to resist Bm CPV infection, making clear the resistance difference between various silkworm strain, and layes foundation for studying on related key genes.