Identification Of Midgut And Peritrophic Membrane Proteins Of Silkworm,Bombyx Mori Interacted With BmNPV

Silkworm(Bombyx mori):A model organism of Lepidoptera insects,which has a long breeding history in China,plays an important role in biological research as well as in silk industry.Bombyx mori nuclear polyhedrosis virus(BmNPV)is the first pathogen discovered in the insect virus and belongs to the type I baculovirus of Alphabaculovirus genus.The characteristics of the disease caused by BmNPV are fast propagation,highly infectious,severly mortal and also difficult to control.BmNPV is most harmful of the three major viral diseases in sericulture which seriously affect and hamper the development of silk industry.The research on the BmNPV infection and the silkworm's antiviral have been the main focus and imerging issue in this field,however,the molecular mechanism is still not entirely clear.Silkworm is mainly infected by BmNPV through oral route,while midgut and peritrophic membrane(PM)form the first line of defence together against the invasion of exogenous microorganisms in silkworm,therefore,it is important to understand virus infection and the silkworm immune defense through the interaction between virus and host from the perspective of midgut and PM.Our previous study screened and identified 12 midgut proteins interacted with BmNPV by Far Western Blot combined with MALDI-TOF-MS mass spectrometry after the midgut proteins separated by one and two-dimensional electrophoresis.These proteins are:60kDa heat shock protein(HSP60),Vacuolar ATP synthase catalytic subunit B(ATP-B),Enolase(En),Actin(Actin),Arginine Kinase(AK),Ribosomal PO protein(RPO),Mitochondrial prohibitin complex protein 2(PHB2),Phosphpglycerate kinase(PGK),Ganglioside-induced differentiation-associate-protein(GDAP),Regucalcin-like isoform XI(RCX1),Calreticulin(CRT)and Vacuolar ATP synthase catalytic subunit A(ATP-A),respectively.To further verify the interaction between these proteins and BmNPV in vitro,we designed specific primers and amplified the corresponding genes.Later,we transformed the recombinant plasmid into E.coli BL21(DE3)after cloned these genes into pET-28a or pGEX-4T-1 and induced with IPTG to express fusion proteins.The expressed proteins purified by affinity chromatography and analysed by Western Blot.We combined Far Western Blot together with the reverse one to verify the interaction between these proteins and BmNPV.To explore the function of PM against BmNPV invasion,we contrasted the different resistant strains of silkworm fed or not fed with BmNPV to observe PM morphologic variation at different time periods using scanning electron microscope(SEM).Extracted PM total proteins,then screened and identified the PM proteins interacted with BmNPV using Far Western Blot combined with LC-MS/MS mass spectrometry,subsequently verifyed the genes at the transcriptional level though RT-qPCR at different resistant strains silkworm P50,A35,BC9 and the group fed with BmNPV.The results are as follows:1.We successfully got the total RNA of midgut and obtained the cDNA.We amplified the 12 genes and sequencing results were consistent with the NCBI published sequence.The proteins were successfully expressed induced by IPTG after successfully constructed recombinant plasmid and the fusion proteins were purified.2.The interaction results indicated that all of the expressed proteins had obvious hybridizing signal bands except AK and RCX1.The results further identified the interaction between BmNPV and these proteins.3.Scanning electron microscopy revealed that PM of P50 began to be damaged at 4h while A3 5 at 6h after fed with BmNPV.The PM of P50 have a large number of holes at 12h while the A35 have only a small amount of perforation at the same time point,which indicated that the PM plays an important role in the BmNPV invasion.4.We obtained 3 proteins in PM interacted with BmNPV,LC-MS/MS showed that these proteins are proteases:triacylglycerol lipase(TGL),serine protease precursor(SPP)and Trypsin-like protease(TLP),respectively.RT-qPCR revealed that the relative expression level of each gene is significantly different in different resistant silkworm strains as well as the BmNPV infected group,respectively.Taking together,our research successfully verified 10 proteins interacted with BmNPV,and preliminarily explored the role of PM in resisting BmNPV,then screened the PM proteins combined with BmNPV as well.These achievements provided reference to understand and analyse the molecular mechanism of BmNPV invasion and silkworm immune response to BmNPV infection.