Study On The Molecular Mechanism Of LncRNA Regulating The Proliferation Of BmNPV

Bombyx mori nucleopolyhedrosis virus(BmNPV)which is highly infectious.Once the silkworm is infected with the virus,it can spread on a large scale within the population,causing a large number of deaths of the silkworm.At present,the prevention and treatment of silkworm virus disease in sericulture industry mainly depended on prevention.A variety of response genes including lncRNAs in silkworm changed when upon virus infection.However,the mechanism of the silkworm lncRNA in the silkworm-virus interaction is still unknown.Therefore,it is necessary to identify and functional analysis of lncRNAs related to BmNPV infection.The result can provide a new theoretical basis for the analysis of the pathogenic mechanism of BmNPV infection of silkworms and the interaction mechanism of silkworm-BmNPV,which is important for the identification and control of silkworm virus diseases.Our study performed the whole transcriptome sequencing to screen and identify lncRNAs related to BmNPV infection and carried out the functional study of partial lncRNAs.The aim was to explore the mechanism of lncRNA in response to BmNPV infection in silkworms by analyzing the spatiotemporal expression profile,and the gain-loss function of lncRNA in the cell on the proliferation of BmNPV,The relevant experimental results are as follows:1.Screening and identification of lncRNA and protein coding genes related to BmNPV infectionThe cells of the silkworm BmN cultured for 0 h and BmNPV infected 72 h were set as the control group(C group)and the experimental group(T group),respectively.The total cellular RNA was extracted for whole transcriptome analysis.The lncRNAs were screened and identified using bioinformatics analysis methods;The differentially expressed(DE)lncRNAs,micro RNAs(miRNAs)and m RNAs were analyzed by DEseq with log2FC>2and padjust<0.05.The target genes(cis-acting and trans-acting)of differentially expressed lncRNA and miRNAs were predicted by bioinformatics software and performed GO and KEGG pathway analysis.The results of DElncRNAs showed that a total of 4450 differentially expressed lncRNAs were screened in BmNPV-infected cells and control cells,including 2837 up-regulated and 1613 down-regulated;The enrichment analysis results showed that target genes of DElncRNA were mainly involved in nucleic acid binding,RNA binding,intracellular organelles,cytoplasmic parts,cellular metabolic processes and gene expression,etc.2.Identification of miRNAs related to BmNPV infection and construction of ce RNA regulatory networkUsing small RNA sequencing,a total of 66 DEmiRNAs were identified in infected BmNPV cells and control cells,of which 35 were up-regulated and 31 were down-regulated.The enrichment pathways of arget genes involved the wnt signaling pathway and ubiquitin-mediated proteins Hydrolysis,lysosome,Jak-STAT signaling pathway,Fox O signaling pathway,etc.Based on the theory of endogenous competitive miRNA,the lncRNA-miRNA-m RNA gene regulatory network related to BmNPV infection in silkworm cells was constructed.The network showed that a total of 1081 down-regulated lncRNAs could act as sponge to bind to 26 up-regulated miRNAs which could target 684down-regulated m RNAs.In addition,1040 up-regulated lncRNAs can interact with 12down-regulated miRNAs.3.Research on the mechanism of Lnc＿209997 in the process of interaction between BmNPV and BmN cellsThe expression characteristics of Lnc＿209997 in various tissues at 72 h of silkworm and fat body at 12-72 h were analyzed by qRT-PCR method.The results showed that the expression levels of Lnc＿209997 in silk gland and fat body were relatively high.The expression level of Lnc＿209997 in body fat upon BmNPV after 72 h was significantly lower than that in the control group(P <0.05).RNA interference technology was used to knock down the expression of Lnc＿209997in silkworm cells,and RT-q PCR was used to analyze the copy number of BmNPV genome in the experimental group and the control group.The results showed that the copy number of the virus genome in the control group was significantly lower than that in the experimental group,indicating that knocking down Lnc＿209997 can significantly promote the proliferation of BmNPV.The Lnc＿209997 gene was amplified and cloned using silkworm c DNA as a template to construct an overexpression vector.it was found that the viral genome DNA content decreased significantly after the recombinant vector was transformed into BmN cells,indicating that overexpression of Lnc＿209997 can inhibit the replication of BmNPV.Subsequently,miR-275-5p could target Lnc＿209997 according to bioinformatics software analsyis.The binding relationship between Lnc＿209997 and miR-275-5p was confirmed by the luciferase reporter gene vector experiment.Overexpression of Lnc＿209997 in cells can inhibit the expression of miR-275-5p,and inhibition of Lnc＿209997 can promote the expression of miR-275-5p.Overexpression of miR-275-5p in cells can promote the proliferation of BmNPV.The results showed that the targeting relationship between Lnc＿209997 and miR-275-5p play a role in the process of BmNPV infection of silkworm.The results of the study are helpful to analyze the mechanism of lncRNA in BmNPV infection,and can provide new clues for elucidating the molecular mechanism of the interaction between the silkworm and the virus.