Studies On The Role Of Bombyx Mori Translation Initiation Factor EIF4E2 In The Infection By AcMNPV

Bombyx mori is an important economic insect in China.Blood sepsis caused by Bombyx mori nucleopolyhedrovirus(Bm NPV)causes great economic losses every year.In order to solve this problem,the researchers launched a silkworm antiviral molecular mechanism research,at present a lot of resistance genes and the resistance mechanism were reported,to clarify the silkworm resistance mechanism laid the important data base,but the related research progress in recent years,with silkworm and Bm NPV as the research object is showing signs of the bottleneck.Autographacalifornica nucleopolyhedrovirus(Ac MNPV)is a baculovirus with high similarity to Bm NPV.Although it has a wide host domain,Ac MNPV can only infect a few silkworm varieties.These results provide a new breakthrough for elucidating the molecular mechanism of resistance to Bm NPV infection.In our laboratory,two kinds of silkworm with different resistance of Ac MNPV were stored as materials,and the transcriptomes of the two strains were compared.Bombyx mori eukaryotic Initiation factor 4E-2(Bme IF4E2)was found.Bme IF4E2 was the most differentially expressed candidate genes related to virus infection.This study aimed to clarify the molecular mechanism of Bme IF4E2’s involvement in Ac MNPV infection,and to provide reference for clarifying the molecular mechanism of Bm NPV infection in Bombyx mori.The specific results are as follows:(1)In order to preliminarily understand the function of Bme IF4E2,the amino acid sequence and phylogenetic tree of Bme IF4E2 were analyzed through bioinformatics.Bme IF4E2 has a high homology with other species e IF4E2 and tends to be conserved evolutionarily.Secondly,the spatiotemporal expression pattern of Bme IF4E2 in Bombyx mori was analyzed,and it was found that Bme IF4E2 was highly expressed in hemolymph of p50,and the highest expression level was found in egg,molting stage and pupa stage.In order to further analyze whether Bme IF4E2 was involved in virus infection,the expression level of Bme IF4E2 was detected in different tissues of silkworm with different resistance before and after virus infection.The results showed that the expression level of Bme IF4E2 was stable after Ac MNPV infection in midgut and malpighiam tube of resistant strain C108,and showed low expression level in fatbody and hemolymph,while the expression level of Bme IF4E2 in p50 was significantly upregulated,suggesting that Bme IF4E2 was involved in the infection process of Ac MNPV.(2)In order to clarify the role of Bme IF4E2 in viral infection,on the one hand,synthetic si RNA was used to conduct RNAi for this gene in Bm N cells,and the influence of interference on the expression of lef3 and GP64 virus protein was analyzed.The results showed that the expression of lef3 and GP64 in knockdown group was significantly lower than that in control group.On the other hand,the repressor 4E2 RCat of Bme IF4E2 was used to inhibit the translation process in which Bme IF4E2 was involved,and the expression of lef3 was consistent with RNAi results.In this study,Bme IF4E2 was overexpressed at the cellular level by constructing p IZT-m Cherry-Bme IF4E2 overexpression vector.The results showed that lef3 and GP64 expression levels in Bm N cells overexpressing Bme IF4E2 were significantly higher than those in the control group.Therefore,Bme IF4E2 can promote the proliferation of Ac MNPV.(3)In order to determine whether Bme IF4E2 participates in the process of infection promotion through interaction with Ac MNPV,the prokaryotic expression vector p ET-28a-Bme IF4E2 was constructed to induce the expression of Bme IF4E2 protein and purify it.The interaction between Bme IF4E2 and Ac MNPV was verified by virus incubation experiment.Results the interaction signals were detected in the experimental group.In conclusion,Bme IF4E2 is involved in the infection process of Ac MNPV,and promotes the virus infection in silkworm cells through interaction with virus.This study provided data reference for elucidating the molecular mechanism of baculovirus infection and theoretical support for efficient development of biological insecticides.