The Mechanism Of Bmiap Gene In BmNPV Proliferation

Silkworm is a fusule insect,and the sericulture is a traditional industry in China.However,the occurrence of silkworm disease has led to a significant decrease in the sericulture production,especially caused by the Bombyx mori muclear polyhedrosis virus BmNPV.Apoptosis is the cell autonomic decease by gene controlling to maintain the homeostasis of cell.It plays an important role in many biological processes,such as the growth and development,organ formation,body shape,and the host immune response.As an effective strategy in the innate immune response in silkworm,an in-depth analysis of the mechanism of apoptosis can promote the further improvement of the mechanism of innate immune response in silkworm,and provide the theoretical basis for cultivation of silkworm antiviral varieties.In this study,the apoptosis related gene Bmiap was cloned and identified,and the mechanism of Bmiap in BmNPV proliferation was analyzed.The results as follows: 1.Cloning,identification and functional analysis of Bmiap gene in silkwormWe successfully cloned the Bmiap gene and obtained a cDNA sequence consisting of its complete ORF.The CDS was 1041 bp in length and encoded 346 aa.The predicted molecular weight was 38.86 kDa and the isoelectric point was 6.49.Bmiap has a conserved C-terminal BIR domain and an N-terminal RING domain.Then,the overexpression and CRISPR/Cas9 knockout vector were constructed.After Bmiap overexpression vector was transfected into BmN-SWU1 cells,immunofluorescence found that the gene was localized in the cytoplasm,and western blot showed that the specific band near 40 k Da was detected.The nucleus shrinks and apoptotic bodies formed,the positive rate of Tunel staining and the activity of caspase increased after CRISPR/Cas9 knockdown,indicated that apoptosis occurs and Bmiap has an effect on inhibiting apoptosis.2.Study on the interaction between Bmiap and BmNPViaps in silkwormThere are two IAP family proteins BmNPViap1 and BmNPViap2 were identified in the BmNPV.In this part,the interaction btween Bmiap and BmNPViap1,BmNPViap2 were identified by immunofluorescence and immunoprecipitation analysis respectively;qRT-PCR analysis showed that Bmiap and BmNPViap2 were down-regulated at 72 h and 48 h after BmNPV post-infection,respectively,while the expression of BmNPViap1 was up-regulated significantly at 72h;After overexpression of Bmiap,there were up-regulated in BmNPViap1 and Vp39 expression,while BmNPViap2 was down-regulated;on the contraty,the expression of BmNPViap1 and Vp39 were down-regulated,and BmNPViap2 was up-regulated after knockout the Bmiap;overexpression of BmNPViap1 and BmNPViap2 increased the expression of Bmiap in BmNPV-infected cells,and Vp39 were up-regulated,whereas there was no significant difference of Vp39 in Bm NPV post-infection 72 h in overexpression of BmNPViap1.These results suggest that Bmiap and BmNPViaps expression showed a trend of increase in the BmNPV early infection,but in the late,Bmiap and BmNPViap2 expression decreased when BmNVPiap1 was further increased;Bmiap can increases the expression of BmNPViap1 and inhibits the BmNPViap2,and promotes the proliferation of BmNPV.Meanwhile,BmNPViap1 and BmNPViap2 can lead the Bmiap up-regulated,and the BmNPViap2 will promote the proliferation of BmNPV,but the Bm NPViap1 had no effect on viral proliferation.3.Study on the mechanism of Bmiap gene in BmNPV infectionIn this part,the interacion protein of Bmiap was screened by using Bmiap as a bait protein by immunoprecipitation.A heat shock protein family member Bmhsc70-4 and a protein phosphatase Bmpp5 were identifited.Immunoflurescence and immunoprecipitation were used to comfirm the interaction between Bmiap and Bmhsc70-4,Bmpp5 were exist,respectively;futher study found that Bmhsc70-4 and BmPP5 all had the anti-apoptosis function;in this study,we found that the mRNA levels of Bmhsc70-4 and Bmpp5 were up-regulated in BmNPV infection;overexpression of Bmhsc70-4 and Bmpp5 promoted the expression of Bmiap and VP39 in infected-cells,while Bmiap and Vp39 were down-regulated after Bmhsc70-4 and Bmpp5 had been knocked out;overexpression of Bmiap,Bmhsc70-4 and Bmpp5 upregulated,knocked out Bmiap,Bmhsc70-4 and Bmpp5 expression down.The above results show that Bmiap and Bmhsc70-4,Bmpp5 have a mutual promotion between the role.