The Study Of The Mechanism Of Bombyx Mori L.Response To BmNPV Infection Based On Transcriptomics And Subcellular Proteomics

The silkworm Bombyx mori L.(Lepidoptera:Bombycidae)is not only an important model organism of Lepidoptera,but also a vital economical insects.Recent years,silkworm industry still plays an important role in the economical development of many developing countries.Bombyx mori nucleopolyhedrovirus(BmNPV)is a primary silkworm pathogen and annually causes serious economic losses.Silkworm midgut is the first barrier to resist the infection of pathogens,thus the study of the first response of silkworm midgut to BmNPV infection will contribute to clarify the molecular mechanism of silkworm resistance to BmNPV infection.In this study,the comparative transcriptomes and subcellular proteomics were used to analyze the alteration in the midgut of BC9 and P50 following BmNPV infection.Besides,Far-western blot was used to identify BmNPV interacting proteins from the subcellular fractions of P50 midgut according to the correlation analysis of the transcriptomics and subcellular proteomics.Based on the study above,the molecular mechanism of silkworm resistance to BmNPV will be disscussed.The main results are as follows:1.The analysis of different resistant silkworm midguts response to BmNPV infection at transcript level based on comparative transcriptomes.A total of 14,300 unigenes were obtained from the transcriptomes of BC9 and P50 following BmNPV infection;of these,869 differentially expressed genes(DEGs)were identified after comparing the four transcriptomes.The number of DEGs between up-regulation and down-regulation did not show difference in P50 following infection and between the two strains,while the number of the down-regulated genes was 2-fold greater than that of up-regualted genes in BC9 following infection.Based on the analysis of GO,the categories of macromolecular complex,transporter activity,biological progresses,and localization related DEGs were significant different in the two strains following infection.Many DEGs associated with protein metabolism,cytoskeleton,and apoptosis may be involved in the host response to BmNPV infection.Moreover,some immunity related genes were also altered following BmNPV infection.Specifically,after removing genetic background and individual immune stress response genes,22 DEGs were found to be potentially involved in repressing BmNPV infection.These genes were related to transport,virus replication,intracellular innate immune,and apoptosis.To further validate the function of these resistant related genes,the expression levels of these genes were analyzed in resistant strains A35,BC9 and susceptible strain P50 using reverse transcription quantitative PCR(RT-qPCR).2.The analysis of different resistant silkworm midguts response to BmNPV infection at protein level based on subcellualr proteomics.To clarify the anti-BmNPV mechanism of the silkworm,the near-isogenic line BC9(resistant strain)and the recurrent parent P50(susceptible strain)were used in a comparative subcellular proteomics study.Two-dimensional gel electrophoresis(2-DE)combined with mass spectrometry(MS)was conducted on proteins extracted from the cytosol,mitochondria,and microsome of BmNPV-infected and control larval midguts.A total of 87 proteins were successfully identified from the three subcellular fractions.These proteins were primarily involved in energy metabolism,protein metabolism,signalling pathways,disease,and transport.In particular,disease-relevant proteins were especially changed in microsome.After infection with BmNPV,differentially expressed proteins(DEPs)primarily appeared in the cytosolic and microsomal fractions,which indicated that these two subcellular fractions might play a more important role in response to BmNPV infection.After removing genetic background and individual immune stress response proteins,16 proteins were identified as potentially involved in repressing BmNPV infection.Of these proteins,the differential expression patterns of 8 proteins according to RT-qPCR analysis were consistent with the 2-DE results.3.The correlation analysis of the transcriptomics and subcellular proteomics.To analyze the molecular mechanism of silkworm resistance to BmNPV infection on a global view,the data of transcriptomics and subcellular proteomics was integrately analyzed.The results showed that transcriptomics and subcellular proteomics could be correlated into many pathways.Besides,V-ATPase family genes were shared by transcriptomics and subcellular proteomics.These genes and proteins will be study deeply in the future.4.Analysis of the interaction between silkworm midgut V-ATPase and BmNPV.The BmNPV interacting protein,V-ATPase A and B,were identified from silkworm midgut mitochondria based on the SDS-PAGE combined with Far-western blot and LC-MS/Ms.To further validate the interaction between V-ATPase A and B with BmNPV,these two genes were expressed in Escherichia coli(E.coli)and then determined the interaction between the fusion proteins and BmNPV using Far-western blot.In this study,many differentially expressed genes and proteins responsed to BmNPV infection were identified from transcriptomics and subcellular proteomics of different resistant silkworm strains following BmNPV infection,respectively.The correlation analysis showed that the transcriptomics and subcellular proteomics could be correlated into many relevant pathways.Besides,V-ATPase A and B have been validated to be interacted with BmNPV.These results provided an overview of silkworm midgut response to BmNPV infection and laid a foundation for clarifying the mechanism of silkworm resistance to BmNPV infeciton.