The Molecular Mechanism Of Midgut Resistant Against BmNPV In Different Resistance Silkworm Strains Based On ITRAQ

The silkworm Bombyx mori is an important economic insect that plays a critical role in silk industry.Furthermore,it is widely used in applied biology as a model organism.However,silkworm is also vulnerable to various pathogenic microorganisms.Among them,the perniciousness of B.mori nucleopolyhedvirus is the most serious,and it causes tremendous loss in sericulture every year.Infection of BmNPV in silkworm is mainly depend on oral infection,and replicates as well as assembles in the midgut columnar epithelial cell.Eventually,the progeny virus will be released and continues to infect other cells.Therefore,the silkworm midgut is the first immune barrier to defence against pathogen infection.In the present study,the silkworm susceptible strain P50 and resistant strain BC9 were used as the experimental materials.Firstly,the resistant difference between the susceptible strain P50 and resistance strain BC9 was examined and determined cross-clamp time by BmNPV,and further screened the differentially expressed proteins(DEPs)in P50 and BC9 after BmNPV infection and the DEPs between P50 and BC9.The candidate protein named ?-1,3-glucan recognition 4(Bm?GRP4)was identified and maybe it is involved in BmNPV resistance.Finally,the correlation analysis of iTRAQ database with transcriptome database was performed.The major research results and conclusions are as follows:1.The analysis of resistant difference in different resistant strain P50 and BC9.Anatomical observation showed that the structure of midgut in BC9 has not been damaged and the hemolymph exhibited yellow transparent,while the midgut was damaged significantly and the hemolymph showed milk white in P50 at the fifth day after BmNPV infection.The analysis of virus copy numbers revealed that virus proliferation in resistant strain BC9 was more than susceptible strain P50 in the midgut-oral infection group and the similar trend was exhibited in the fat body.In the midgut-hypodermic infection group,the virus copy numbers in two silkworm stains had no significant difference.2.iTRAQ-based quantitative proteomics analysis of the molecular mechanisms of Bombyx mori larval midgut response to BmNPV in different resistant strains.iTRAQ analysis was conducted by using resistant silkworm strain BC9 and susceptible silkworm strain P50.A total of 793 DEPs were identified from different comparable groups.GO and KEGG analysis showed that these DEPs were mainly involved in metabolism,catalytic activity,amino sugar and nucletic sugar metabolism and carbon metabolism.Among them,114 DEPs were identified related to cytoskeleton,immune response,apoptosis,ubiquitination,translation,ion transport,endocytosis and endopeptidase activity.After removing genetic background and individual immune stress response DEPs,84 DEPs were found to be potentially involved in repressing BmNPV infection.3.Identification and function analysis of B.mori midgut pattern recognition receptor?-1,3-glucan recognition protein 4(Bm?GRP4).Based on above iTRAQ results,we screened Bm?GRP4 according to bioinformatics analysis.RT-q PCR and western blot results revealed that Bm?GRP4 had a high expression in the midgut and malpighian tubule,while it showed low expression in other tissues.What's more,the relative expression level of Bm?GRP4 was up-regulated after BmNPV infection.Injection of anti-Bm?GRP4 serum experiment further suggested that Bm?GRP4 was likely to involve in repressing BmNPV infection.4.The correlation analysis of the quantitative proteomics iTRAQ and transcriptomics revealed the molecular mechanisms of silkworm midgut response to BmNPV.Correlation analysis of the iTRAQ with transcriptome suggested that amino sugar nucleotide sugar metabolism and lysosome were found in the P50+ vs.P50-and BC9+ vs.BC9-.In the BC9-vs.P50-,the protein processing in endoplasmic reticulum was identified.In conclusion,this study revealed the molecular mechanisms of silkworm larval midgut response to BmNPV based on quantitative proteomics iTRAQ,and identified the DEPs response to BmNPV comprehensively and analyzed their functions.Eventually,the possible molecular mechanisms of silkworm larvae response to BmNPV were analyzed according to relevant metabolism pathways from correlation analysis.