Functional Research Of A Peritrophin Gene Bm011851 During BmNPV Infection In Bombyx Mori

Bombyx mori as a representative of lepidoptera insects,has important economic value.The growth and development of Bombyx mori will be threatened by various pathogenic microorganisms,among which Bombyx mori nuclear polyhedrosis virus(BmNPV)causes 70% of the Bombyx mori diseases,which has a serious impact on the sericulture production.Peritrophic membrane(PM)is the first line of defense for insects to resist the invasion of exogenous pathogens.Peritrophins are closely related to the invasion of insect midgut cells by exogenous pathogens.In this study,peritrophin gene Bm011851 of Bombyx mori was taken as the research object,the gene Bm011851 was cloned and analyzed.To obtain and detect the growth and development,economic characters and their effects on the surface structure of PM in Bombyx mori larvae of overexpression Bm011851 transgenic Bombyx mori.The function of Bm011851 in the process of BmNPV infection of Bombyx mori larvae was studied to analyze the mechanism of Bm011851 participating in the resistance to virus invasion.The main research results are as follows: 1.Cloning and expression analysis of the peritrophin gene Bm011851 of Bombyx moriGene expression analysis showed that Bm011851 was expressed in Bombyx mori larval stage,the expression level was higher in feeding stage than that in sleeping stage,the expression level of 5th instar larvae reached the highest level,the expression level of larvae on 6 days of 5th instar began to decline.Bm011851 was highly expressed in the midgut,and the expression levels in the middle part of the midgut and posterior midgut were higher than that in the anterior midgut,and almost no expression in the foregut,hindgut and carcass.The cloning results showed that the ORF of Bm011851 was 1620 bp in length and encoded 539 amino acids.The encoded protein sequence had no O-linked glycosylation site and N-linked glycosylation site,contains one transmembrane domain and one N-terminal signal peptide.After oral infection with BmNPV in 4th instar larvae,Bm011851 expression in midgut tissues was significantly up-regulated at 3 h,6 h,12 h,18 h,24 h,30 h,48 h,78 h and 84 h.2.Acquisition of transgenic Bombyx mori with overexpression Bm011851The transgenic overexpression vector of pBac[3xp3-dsred-sv40 +opie2-Bm011851-v5-his-OpIE2_pA] was constructed,microinjection of bombyx mori eggs,and the positive Bombyx mori was screened.Molecgical biological tests showed that Bm011851 was successfully overexpressed in Bombyx mori.The weight of the larvae,total cocoon weight,cocoon layer quantity and cocoon layer rate of Bombyx mori were counted,and it was found that the overexpression of Bm011851 did not affect the normal growth and development of Bombyx mori and its main economic traits.Sem observation showed that transgenic overexpression of Bm011851 did not directly effect on the surface structure of PM.The rusults of qRT-PCR showed that the expression of other peritrophins genes Bm001504,Bm009641,Bm001491,and Bm000185 in transgenic Bombyx mori had no significant changes compared with the control.The results showed that the transgenic Bombyx mori could be used for functional study of Bm011851.3.Functional research of Bm011851 during BmNPV infection in Bombyx moriThe investigation found that the transgenic overexpression of Bm011851 could improve the survival rate of Bombyx mori after BmNPV infection,and the half lethal dose of BmNPV was increased 10 times compared with control.Overexpression of Bm011851 could inhibit the expression of the essential early gene(ie1),intermediate gene(vp39)and late gene(p10)for the proliferation of BmNPV.Sem was used to observe the surface structure of PM at different time points after BmNPV infection in transgenic Bombyx mori.It was found that overexpression of Bm011851 could reduce the damage degree of PM caused by BmNPV,which was conducive to maintaining the integrity of PM.Observation of midgut tissue section found that Bm011851 could reduce the damage of midgut cells during BmNPV invasion of Bombyx mori,maintain the stability of midgut cells.Peritrophins may affect the infection of BmNPV on Bombyx mori larvae by inhibiting virus replication and affecting the integrity of PM and the stability of midgut tissue cells.The mechanism needs further study.