Analysis Of MicroRNA And Its Target Genes Related To Nucleopolyhedrosis Virus Infection In Silkworm, Bombyx Mori

Bombyx mori nuclear polyhedrosis virus(BmNPV)is one of the major pathogens in the sericulture industry.microRNAs(miRNAs)are widely involved in a series of important life processes including growth and development,apoptosis,and resistance to viruses,and play an important role in the interaction between host and pathogenic microorganisms.In this study,Solexa high-throughput sequencing technology was used to perform small RNA sequencing analysis on BmNPV-infected silkworm and control silkworm tissues for 72 hours.Real-time fluorescence quantitative PCR analysis was used to verify differentially expressed miRNAs.Bioinformatics software was used to predict their target genes and their biological functions.This experiment selected one of the newly discovered differentially expressed miRNAs named miR-277-5p for further study.A dual-luciferase reporter vector assay system was used to verify that miR-277-5p can target the silkworm methyltransferase gene(Dnmt).the silkworm BmN cells were treated with the methylase inhibitor Zebularine and infected with BmNPV.At the same time,The target gene expression was silenced by the crispr/cas9 technique.To explore the futher effect of silencing the target gene on the proliferation of the virus.The relevant experimental results are as follows:1.Solexa sequencing analysis of differentially expressed miRNAsThrough the small RNA sequencing analysis of the infected group and the control group,the miRNA expression profile was initially revealed.A total of 373 known Bombyx mori miRNAs and 73 new miRNAs were obtained,of which 37 new miRNAs were conserved.A total of 38 differentially expressed miRNAs were detected,of which 23 were up-regulated and 15 were down-regulated.Ten randomly selected miRNAs were selected and verified by quantitative stem-loop PCR.The results showed that 9 of the differentially expressed miRNAs were consistent with the solexa sequencing results.GO analyses differentially expressed miRNA predicted target genes.More than 50% of target gene function involves binding,catalysis,metabolism,and the like.Nearly 6% of target genes are involved in viral particles,immune processes and stress responses.2.Prediction and verification of miR-277-5p target genesThe expression of miR-277-5p was significantly up-regulated in silkworm infected.The use of bioinformatics software predicts that methyltransferase 2(Dnmt2)is one of the target genes of this miRNA and that it acts on the 3' UTR.The luciferase reporter genevector experiments confirmed the targeting effect of the miRNA on Dnmt2.3.The role of Dnmt gene in silkworm-BmNPV interactionUsing the bioinformatics software to design the gRNA of the conserved region of the Dnmt gene sequence of Bombyx mori,the CRISPR/Cas9 triple plasmid,as CRISPR/Cas9-Dnmt,was successfully constructed.BmN cells were transfected with the constructed plasmid and infected with BmNPV.Cell genomic DNA was extracted and cloned and sequenced to verify whether the target gene was mutated.According to the sequencing result and the original sequence alignment,it was found that there was a significant deletion and insertion,indicating that the Dnmt gene mutation was successful.1)On one hand,treatment of BmN cells with the methylase inhibitor Zebularine and infection with BmNPV-GFP showed that the proliferation of BmNPV in the experimental group was significantly lower than that of the control group.The genomic DNA of the cells was extracted,and the ie-1 gene of BmNPV was detected by fluorescence quantitative PCR.The results showed that compared with the control group,the proliferation of BmNPV was significantly decreased in the BmN cells treated with Zebularine.2)On the other hand,BmN cells were transfected with the constructed CRISPR/Cas9-Dnmt vector and a control,and BmNPV-GFP was infected.Fluorescence showed that the proliferation of BmNPV in the experimental group was significantly lower than that in the control group.The genomic DNA of the cells was extracted,and the ie-1gene of BmNPV was detected by fluorescence quantitative PCR.The results further confirmed that the BmNPV proliferation level decreased after Dnmt mutation.The above experimental results show that Dnmt from Bombyx mori plays an important role in the proliferation of BmNPV.The above findings provide new clues for our in-depth study of the interaction mechanism between BmNPV and BmNPV,providing a theoretical basis for further investigation of epigenetics in the regulation of silkworm virus infection,and providing a new basis for the development of biological control of BmNPV.