Functional Analysis Of Bombyx Mori Insulin-related Peptide Binding Protein 2 And Identification Of Its Interaction Proteins In Silkworm

Silkworm,Bombyx mori,is a model organism of Lepidoptera.It is also an important model for genetic research.Virus disease of silkworm is one of the major reason which always cause enormous damage to the sericultural industry for a long time.Bombyx mori cytoplasmic polyhedrosis virus(Bm CPV)is one of the major viral pathogens for the silkworm.It specifically infects the epithelial cells in the midgut of silkworm larvae.However,there are few reports about the machnism of silkworm in defensing BmCPV infection.Prevention is the main measures for control of the BmCPV caused silkworm disease in sericultural industry.Different silkworm strains have different resistances to BmCPV.But we have not yet successfully cultivated any anti-BmCPV silkworm strain at present.With the goal of obtaining an overall view of the molecular mechanism of Bm CPV invasion and antiviral immunity of different silkworm strain,high-throughput sequencing was performed in our previous study to investigate the gene expression profiles of 4008(susceptible to BmCPV)and P50(resistant to BmCPV)silkworm strain after oral infection with BmCPV.The expression level of Bombyx mori insulin-related peptide-binding protein2(BmIBP2)was identified to be significantly up-regulated in the midgut of BmCPV-infected silkworms,and the up-regulation was higher in the resistant silkworm strain than the susceptible strain.BmIBP2 may play an important role in the biological metabolic regulation,apoptosis,viral resistance of invertebrates and it may be involved in the defence mechanism of silkworm against BmCPV-infection.In the present study,we expressed successfully BmIBP2 protein and explored its effects on BmN cell proliferation.Then,we studied the function of BmIBP2 with RNA interference and over-expression techniques.Furthermore we screened and obtained an interactive protein with BmIBP2 in the BmN cells.The major results are as follows.1.The differential expression of BmIBP2 in midgut of silkworm larvae infected with BmCPV and other pathogensAfter the silkworm larvae were infected by the pathogens-BmCPV,Bombyx mori nuclear polyhedrosis virus(BmNPV),Bombyx mori bidensovirus(BmBDV),Beauveria bassina,Nosema bombycis,the expression profiles of BmIBP2 in midgut at different time points were detected by quantitative real time PCR(qRT-PCR).The results suggested thatthe expression of BmIBP2 is not only up-regulated in the BmCPV infected midgut of silkworm larvae,but differential expression was detected in the midgut infected with the tested other pathogens.It is implied that BmIBP2 might be involved in the process of silkworm response to pathogen infection and play an important role in silkworm immune defense.2.Effects of BmIBP2 protein on BmN cell proliferationBmIBP2 protein was expressed by the prokaryotic expression system.The influence of BmIBP2 protein on cells proliferation was determined by adding the purified and renatured protein into BmN cell cultures and by MTT method.Then the regulation of hydroxyecdysone 20 E on BmIBP2 expression was also validated.The results showed that the purified protein had a certain inhibitory effect on silkworm BmN cell proliferation and20 E could promote the expression of BmIBP2 genes.The function of BmIBP2 was further analysised by the technology of RNAi and overexpression.We transfered the chemically synthesized siRNA and negative control siRNA of BmIBP2 into the BmN cell,then tested the cell proliferation.At the same time,we detected the changes in the expression of BmIBP2 and respiratory enzyme genes related to glycometabolism.As the result of RNA interference,the expression of BmIBP2 was down-regulated campared with the control groups.The speed of the cell proliferation of BmN cell became faster and the expression of the respiratory enzyme genes related to glycometabolism were up-regulated after the BmIBP2 was silenced by RNAi.On the contrary,we establish a transgenic expression vector pIZT-IBP2 and transfected it into Bm N cells to overexpress BmIBP2,as a result the speed of the BmN cell proliferation became slower as compared with control groups,and consequently the expression of respiratory enzyme genes related to glycometabolism presented down regulation.3.Screening and identification of proteins interacting with BmIBP2 proteinThe BmIBP2 protein fused with enhanced green fluorecence protein(EGFP)was expressed with Bac-to-Bac baculovirus expression system in BmN cells.The EGFP was reporter gene.The recombinant baculovirus shuttle palamid vBmEGFP-IBP2 and vBmEGFPwere constructed by inserting BmIBP2 and EGFP genes into pFastBac1 vector and transform DH10 Bac competent cells.Then the shuttle vectors were transfected into BmN cells to construct recombinant baculovirus.In the transfected BmN cells,fluorescence was observed and a fusion protein of about 55 kDa was detected by SDS-PAGE and Western-Blot analysis.Then the expressed recombinant protein was collected and used to screen proteins interacting with BmIBP2 from the host cells by co-immunoprecipitation method.Aputative protein was obtained and identified by Mass analysis to be Bombyx mori ATP synthase(BmATP).Then yeast two-hybrid analysis confirmed the interaction between BmIBP2 protein and BmATP.Our study results provided a good basis not only for further understanding the roles and mechanism of BmIBP2 involved in silkworm defense against BmCPV,but also for further revealing the biological function of BmIBP2 and the molecular mechanism of silkworm resistance to BmCPV infection.