Studies On Genes Regulating Hormone Levels In The Process Of Infection Of BmCPV In Silkworm

Bombyx mori cytoplasmic polyhedrosis virus（BmCPV）is one of the main pathogens of sericulture.As the silkworm and virus genome sequences are sequenced one by one,the silkworm,a lepidopteran insect,naturally becomes a biological model for studying the interaction between insects and pathogen infections.In order to screen the genes and proteins of anti-BmCPV virus,the early laboratory analyzed the midgut of silkworm by digital gene expression profiling（DGE）and isobaric tags for relative and absolute quantification（iTRAQ）.It was found that multiple hormone-related genes and proteins were differentially expressed after infection with BmCPV.20-hydroxyecdysone（20E）and juvenile hormone（JH）are the two major hormones in metamorphosis insects,which synergistically regulate the molting and metamorphosis of silkworm.This thesis studies whether there is an interaction between hormones and viruses and studies the immune defense mechanism played by hormones in the antiviral process of silkworm,mainly from three aspects:（1）Determination of hormone titer in hemolymph after infection of BmCPV in silkworm（2）Screening differentially expressed genes related to hormone regulation after infection of BmCPV in silkworm;（3）Function of differentially expressed genes related to hormone regulation.The experimental results are as follows:1.Detection of hormone titer in hemolymph after infection of BmCPV in silkwormHigh performance liquid chromatography-ultraviolet（HPLC-UV）method was used to detect the titer of 20E and JH III in the hemolymph of the experimental group and the control group after infection with BmCPV.The average concentration of 20E in the hemolymph of the control group was 1.0916μg/mL,and the average concentration in the experimental group was 0.3926μg/mL.The concentration of 20E in the hemolymph of the silkworm infected with BmCPV virus was about 1/3 of that in the normal silkworm.It indicated that BmCPV virus reduced the concentration of ecdysone and hindered the expression of ecdysone in the body after infection.JH III was not detected because the concentration was too low.2.Screening and identification of hormoner egulatory related genes in Bombyx mori infected with BmCPVBioinformatics methods to screen differentially expressed genes and proteins in the regulation of hormone expression,and further validate differentially expressed genes related to BmCPV infection in silkworm by fluorescence quantitative PCR（qRT-PCR）,predict which signaling pathways these genes pass through.Resist the pathogen and exert molecular immune mechanisms.The results showed that the 5^th instar silkworm infected with BmCPV,compared with the control group,the genes in the 20E synthetic pathway Promoted Prothoracicotropic hormone（PTTH）,Cyp450 family enzyme,Neverland（Nvd）,3dehydrogenated-3β-reductase（3DE-3β-reductase）were down-regulated and then up-regulated in the early stage after infection with BmCPV,which reduced the 20E synthesis in the silkworm,and the titer of 20E in the hemolymph of the 72 hpi silkworm was lower than that of the normal silkworm.In the middle and late stage of BmCPV infection,the up-regulated expression of PTTH,Cyp315A1,Cyp314A1 and Nvd in the 20E synthetic pathway and down-regulated expression of the genes in the metabolic pathway increased the 20E titer in the silkworm,suggesting that it is involved in apoptosis and exerts antiviral effect.The down-regulated expression of the Ecdysone receptor（EcR-A）,Ultraspicacle（USP）,Broad-complex（Br-C）and Nuclear hormone receptor（E75）in the 20E signaling pathway blocked the cascade reaction of 20E and delayed the molting time.The genes of JH synthesis pathway were mainly up-regulated expression,and the genes of JH esterase（JHE）,JH epoxide hydrolase（JHEH）in the metabolic pathway were mainly down-regulated expression.It were speculated that the concentration of JH increases during the infection of the virus,prolongs the larval stage,and delays the sleep time.JH binding protein（JHBP）、Krüppel homolog 1（Kr-h1）were up-regulated in the signaling pathway.These hormone-regulated genes were differentially expressed after BmCPV infection in silkworm,and played a role in the process of resisting BmCPV in silkworm,and participated in the regulation of immune molecules.3.Identification and functional analysis of the new gene FAMeT-2FAMeT-2（BGIBMGA002314）is a key gene in the JH synthesis pathway.In the middle and late stages of BmCPV infection in silkworm,FAMeT-2 was up-regulated in the midgut of silkworm,both in mRNA transcriptionp and poteomics.Therefore,FAMeT-2 was further studied.Tissue differential analysis of the FAMeT-2 gene by quantitative PCR revealed that FAMeT-2 was highly expressed in the midgut of the silkworm.Bioinformatics analysis of the gene showed that the conserved domain DM9 can specifically recognize foreign microorganisms such as bacteria and fungi,and participate in innate immunity through the Imd signaling pathway.The prokaryotic expression vector pET28a-FAMeT-2was constructed,and IPTG induced E.coli expression,and a 27 kDa target protein was obtained.siRNA of FAMeT-2 was synthesized,pIZT-FAMeT-2 overexpression vector was constructed,and transfected into BmN cells of Bombyx mori,respectively.After interference or overexpression of FAMeT-2,total RNA was extracted.Reverse transcription was used as a template to detect cell proliferation,apoptosis and transcription of immune-related genes by quantitative PCR.After RNAi FAMeT-2,the cell proliferation-related genes were down-regulated and the immune pathway PGRP-LB was up-regulated.After overexpression of FAMeT-2,the cell proliferation-related genes were up-regulated and PGRP-LB was down-regulated.It indicated that the change of FAMeT-2 expression not only affected the metabolism and proliferation of cells,but also affected the PGRP-LB to recognize and degrade peptidoglycan,and indirectly participate in the immune regulation process of silkworm.In summary,BmCPV virus infection affected the normal synthesis and metabolism of hormones in silkworm,and resulted differential expression of hormone-regulated genes at the transcriptional level.FAMeT-2 can promote cell proliferation to a certain extent;PGRP-LB specifically recognized and degraded peptidoglycan,participated in innate immunity in Imd signaling pathway.Whether its up-regulated expression after BmCPV infection was involved in the anti-BmCPV defense mechanism of silkworm remains to be further studied.The results of this study not only help to better understand the relationship between viral infection and host hormone regulation mechanism,but also provide a strong basis for understanding the biological functions and antiviral defense mechanisms of silkworm hormone regulation related molecules.