Identification Of Collagenase And Chymotrypsin-like Serine Protease And Differential Expression Analysis Response To BmNPV Infection In Silkworm,Bombyx Mori

Serine proteases are an important class of proteases in lepidoptera insect digestive system and they play important roles in dietary protein digestion,coagulation,signal transduction,hormone activation,inflammation and development.Bombyx mori nuclearpolyhedrosisvirus(BmNPV)is one of the most serious pathogens in sericulture and causes huge economic loss.The molecular mechanism of BmNPV infection and the antiviral of mechanism silkworm has always been a research hotspot and focus.However,the molecular mechanism has not been fully elucidated yet.Based on the results of quantitative proteomics Label-free data of silkworm midgut digestion juice of different resistant strains of silkworm in our laboratory,we successfully screened two differentially expressed candidate proteins B.mori serine proteases collagenase(Bmcoll)and chymotrypsin-like serine protease(Bmclsp).In this study,the serine protease genes Bmcoll and Bmclsp were cloned from the midgut and their sequence structure characteristics and expression characteristics were also analyzed.Additionally,analysis of Bmcoll and Bmclsp expression level at transcriptional level and protein level were analyzed in different resistant strains after BmNPV infection.The main results are as follows:1.Screening of candidate proteins Bmcoll and Bmclsp and verification of transcription levels.Based on the analysis of Label-Free data,the differentially expressed proteins Bmcoll and Bmclsp before and after infection with BmNPV in different resistant strains were obtained,and their results were verified by RT-q PCR.The results showed that the expression levels of Bmcoll and Bmclsp are basically consistent with Label-Free.2.Bioinformatics analysis of Bmcoll and Bmclsp.Bioinformatics analysis showed that there is a signal peptide sequence in Bmclsp protein,which can guide the protein to be secreted extracellularly.What’s more,both Bmcoll and Bmclsp have the trypsin-like serine protease domain(Trypsin-like serine protease)which possess a triplet catalytic center His(H)-Asp(D)-Ser(S)unique to serine proteases.These sequence analysis results provide direction and basis for subsequent research.3.Spatiotemporal expression analysis of Bmcoll and Bmclsp.The semi-quantitative and RT-q PCR methods were used to analyze the spatial and temporal expression of Bmcoll and Bmclsp.The results showed that: Bmcoll and Bmclsp genes were highly expressed in the fifth instar larval stage of silkworm,and lowly expressed in egg,pupa,and adult stages;and highly expressed in the midgut,lowly expressed in other tissues;and their expression levels depended on the changes of hormone.4.Expression analysis of Bmcoll and Bmclsp in response to BmNPV infection in different resistant silkworm strains.The expression analysis of Bmcoll and Bmclsp in different resistant silkworm strains in response to BmNPV infection was analyzed in the resistant strain A35 and the susceptible strain P50.The results showed that the expression levels of Bmcoll and Bmclsp were both higher in resistant strain than in susceptible strain.5.Expression analysis of Bmcoll and Bmclsp protein levels in response to BmNPV infection in different resistant silkworm strains.Prokaryotic expression of Bmcoll and Bmclsp was performed to obtain recombinant proteins and polyclonal antibodies were prepared.The Western Blot method was used to analyze the protein levels of Bmcoll and Bmclsp before and after BmNPV infection.The results showed that:(1)Compared to the control group,the expression level of Bmcoll in the resistant strain A35 was significantly higher than in the sensitive strain P50;(2)Compared to the control groups,the expression level of Bmclsp was up-regulated in the sensitive strain P50 and down-regulated in the resistant strain A35.In summarythe two differential expressed silkworm digestive juice proteins Bmcoll and Bmclsp after BmNPV infection were identified and analyzed.These results indicated that these two proteins have the characteristics of responding to BmNPV infection.The molecular mechanism of the silkworm’s immune response to viral infection provides a basis.