| Silkworm is a kind of important economic and model insect, and it is also a considerable edible resource. With the improvement of living standard, silkworm is generally paid attention due to its high quality of protein and fatty acid, and it is developed to health food to the market successfully. So new quantity and quality demands are set for the sericulture production. In sericulture, the disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral infectious disease, which is difficult to control, and bring about60%loss of production in major sericultural areas of the world. In addition, some recent studies showed that the NPV can entry into mammalian cells with high efficiency, which bring some potential safe problems when the silkworm is used as food resource. So the identification of resistance gene and elucidation of the resistance mechanism are important work. Our group identified a local resistant strain NB from344silkworm lines and constructed a BmNPV-resistant near-isogenic line (NIL) BC8. Using this model, some molecules related to anti-BmNPV were discovered in DNA, RNA and protein levels, but the resistant gene is still unknown.In this study, a method of the second-generation sequencing technology combined bulked segregant analysis (BSA) was employed to analyse the molecular markers and linkage analysis of resistance gene, and the rough location of resistance gene was obtained. Some possible resistance genes or related gene were analysed further.The major findings are as fowllows:1. The BmNPV resistant strain NB and susceptive strain306were reduced-representation sequenced by the second-generation high-throughput sequencing.5441molecular markers (SLAF marker) between NB and306were acquired, consisting of4765SNPs,355EPSNPs and311INDEL.2. Through BLAST with the gene and EST libraries of silkDB using5441molecular markers,327markers were found located in the coding sequence (CDS) after corrected manually item by item,138of which lead to mutation in protein level. These mutated proteins were annotated by COG, GO and KEGG database and found that some proteins involved in the innate immune system, such as serine protease (BGIBMGA006406), serpin (BGIBMGAO13848), chymotrypsin (BGIBMGA008242) and a human HSP40/ DnaJ homologous members (BGIBMGA013135) and two proteins related to DNA replication and repair (BGIBMGA001493, BGIBMGA003650) were mutated in306, suggesting that the sensitive to a variety of pathogenic microorganisms and environment of306, is related to its mutated proteins in innate immune system.3. The BSA (bulked segrgant analysis) method was employed to analyse if the maker is linked to BmNPV resistance.6polymorphism markers were confirmed to link to the BmNPV resistance genotype, and4of which were located in the chromosome23. Finally, a0.67Mb region in chromosome23were considered to be the hot region of BmNPV resistance gene, which contain22predicted genes. Through RT-PCR analysis, the expression of6predicted genes could be detected in midgut of silkworm, and the sequencing data showed two carboxylesterase genes were mutated in NB.4. V-ATPase assay of midgut from NB,306and BC8after inoculated with BmNPV showed that V-ATPase activity were up-regulated in the midgut column cells of BmNPV-resistant strains NB and BC8. Besides, the transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm BmN cells could significantly inhibit BmNPV proliferation, indicating that V-ATPase is involved in the silkworm defense response against BmNPV. |
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