Identification Of Bombyx Mori Peritrophins And Its Function In BmNPV Invasion

The midgut of insects contains a layer of semipermeable membrane with a non-cell structure of elasticity and toughness.The peritrophic membrane?PM?is secreted by the midgut epithelium and forms a sleeve-like structure throughout the midgut area.Insect PM structure has a complex structure and a diverse components in which the protein is the main component.A PM protein that contains a chitin binding domains at least and can be removed by a strong denaturant?urea,guanidine hydrochloride,etc.?,is called Peritrophins.It has been reported that Peritrophins plays an important role in resisting the invasion of exogenous pathogens.In this study,11 Peritrophins were identified by homology search and structural search in the whole genome database of silkworm,and phylogenetic analysis was carried out.Two Peritrophins Bm001504 and Bm009641,that were specifically expressed in the midgut,were screened to make a furture study by phylogenetic analysis and spatio-temporal expression analysis.The experiment was used to analysis the expression levels of two genes in midgut tissues at different time points after BmNPV infection,results showed that Bm001504 and Bm009641 responded to the induction of BmNPV.And then constructed the overexpression vectors and knockout vectors of two genes.The effects of two genes on viral proliferation and replication were examinsed at cellular level.At the same time,the effect of BmNPV on the transgenic silkworm was detected after knockout and overexpression of Bm001504 gene at individual level.The results are as follows:1?Identification and bioinformatics analysis of Peritrophins in Bombyx moriIn this study,40 homologous genes were identified by homology search and domain search in the whole genome database of Bombyx mori.In addition to 3 chitinases containing Glyco_hydro₁8 conserved domain,4 intestinal mucins containing mucin domain,3 chitin deacetylases containing Polysacc_deac₁ conserved domain,13 CPAP1 containing 1 chitin binding domain,6 CPAP3 containing 3 chitin binding domains,and finally got 11 Peritrophins genes.Through the phylogenetic analysis,it was found that the different CBDs of the same gene were clustered.Compared with CBDs of peritrophins in Tribolium castaneum by the multiple sequence alignment,we found that the number of aminso acids among the cysteine residues conform to:CX11-17CX5CX9-14CX12CX6-7C.2?Expression of Peritrophins in Bombyx moriUsing existing chip data,we found that Bm001504,Bm009641 and Bm011851 were especially expressed in the midgut of the larval stage,and the expression of them were significantly up – regulated after oral infection by BmNPV.The expression of peritrophins were analyzed by RT-PCR,the res?Lts were consistent with the chip data.We speculated that Bm009641 and Bm001504 genes are involved in BmNPV infection and are listed as the target gene for study.Firstly,the full length of the two genes was cloned.Both genes were found to contain the signal peptide and the N-and O-linked glycosylation sites.After oral infecting BmNPV,qRT-PCR analysis of silkworm Bm001504 and Bm009641 genes showed Bm001504 gene was up-regulated in 6h,8h,12 h,24h and 72 h after BmNPV infection,Bm009641 gene was up-regulated in BmNPV6 h,8h and 24 h after infection BmNPV.This indicates that the two genes may have important functions in the invasion of BmNPV..3?Study on the Function of Peritrophins in Bombyx moriThe overexpression vector and knockout vector of Bm001504 and Bm009641 genes were constructed respectively.The effects of overexpression two genes on the proliferation and replication of the BmNPV were observed at the cellular level.The results showed that the expression levels of ie1,vp39 and p10 were significantly decreased in the treatment group compared with the control group.It showed that Bm001504 and Bm009641 genes could inhibit the proliferation of the BmNPV at the cellular level.At the individual level,Bm001504 was overexpressed and knocked out respectively,we detected the effect of BmNPV on transgenic Bombyx mori and the survival rate of silkworm larvae.The results showed that the survival rate of silkworm larvae after BmNPV infection was also increased,and the expression levels of the key genes of viral proliferation?ie1,vp39 and p10?were significantly decreased after overexpressing Bm001504.After adding BmNPV,the proliferation of key genes in BmNPV?ie1,vp39 and p10?was significantly increased by knocking out Bm001504,and the survival rate of transgenic silkworm larvae after BmNPV infection was also decreased.The results showed that the Bm001504 and Bm009641 gene play an important role in the protection of silkworm from exogenous pathogenic microorganisms,but their mechanism was to be studied later.