Preliminary Study On The Role Of DNA Methylation In The Replication Process Of Silkworm Nuclear Polyhedrosis Virus

Bombyx mori nucleopolyhedrosis virus(BmNPV)can cause blood type sepsis in silkworm,which seriously endangers the sustainable development of sericulture.DNA methylation is a kind of gene expression regulation which widely exists in epigenetics,and plays an important role in a series of life processes such as embryo development,genome imprinting,aging,and virus infection.So far,there are few reports on the epigenetic regulation of silkworm virus disease at home and abroad.In this study,the methylase inhibitor Zeb(Zebularine)was used to inhibit the DNA methylase(Dnmt)activity of silkworm cells to explore the effect of DNA methylation on the expression of immune-related genes and the proliferation of BmNPV.Further,whole-genome methylation sequencing and transcriptome sequencing were used to compare and analyze the changes in the genomic DNA methylation level and gene m RNA expression level of the infected BmNPV silkworm and the control silkworm,Genes with differences both in m RNA level and DNA methylation level were screened to explore the role of DNA methylation in the interaction between silkworm and BmNPV.The results of the study are as follows:1.The effect of DNA methylase on the proliferation of BmNPVSilkworm cells can induce Dnmt gene expression after 48-96 h infection with BmNPV.The silkworm cells in the experimental group were treated with BmNPV and Zeb(Zeb+BmNPV)at the same time,and the control silkworm cells were treated with BmNPV and DMSO(DMSO+BmNPV)at the same time.Virus titer,Western blot and q RT-PCR were performed on the cells of experimental group and the control group,respectively.The results of the study showed that the inhibition of methylase activity by Zeb significantly reduced BmNPV titer,the protein expression level of BmNPV nucleocapsid protein 39(VP39)and the gene copies of BmNPV ie-1 and lef-3 gene.In addition,Zeb also can repress the expression of iap1 gene.It suggested that inhibiting DNA methylase can inhibit the proliferation of BmNPV.2.The effect of BmNPV infection on the midgut transcriptome of Bombyx moriTotal RNA from the midgut of silkworm infected with BmNPV for 72 h and control samples were extracted and then performed RNA-seq assay.The results showed that after the silkworm was infected with BmNPV,the silkworm transcriptome expression pattern changed significantly.A total of 2,171 differentially expressed genes were detected in silkworm infected midgut tissues,including 1251 down-regulated genes and920 up-regulated genes.The results of GO enrichment showed that the expression levels of genes related to catalysis and metabolic functions were mostly down-regulated;while the expression levels of genes related to DNA binding,membrane binding and chromatin binding functions were mostly up-regulated.The differential expression of the 6 genes was verified by q RT-PCR.3.The effects of BmNPV infection on the whole genomic DNA methylation level of Bombyx moriWGBS(whole genome bisulfite sequencing)was performed on the midgut of the silkworm infected with BmNPV and the midgut of the normal silkworm.Sequencing results showed that the average methylation rate of C bases in the midgut of the silkworm was 0.185%,99% of which were in CG mode.The average methylation rate of CG sites was 0.875%.The DNA methylation levels on different genomic elements from high to low are exons,introns,promoters,and repetitive sequence regions.A total of 241 differentially methylated regions(DMR)were detected in the midgut tissues infected with BmNPV,of which 126 were hypermethylated and 115 were hypomethylated.A total of 177 differentially methylated genes(DMG)were detected.KEGG pathway analysis showed that differentially methylated genes are involved in spliceosome,RNA transport,purine metabolism,lysosome,protein processing in the endoplasmic reticulum,etc.4.Double differential gene screening and verificationThrough comprehensive analysis of the results of whole-genome methylation sequencing and transcriptome sequencing,we found that there are 26 genes that have significant differences both at the transcriptome level and DNA methylation level.The double difference between the chromatin structure maintenance gene(BGIBMGA014008)and the actin 6A gene(BGIBMGA009805)was verified by using q RT-PCR and BS-PCR technology.The verification results showed that part of the gene body regions of the two genes showed high methylation.Its m RNA levels are significantly up-regulated after BmNPV infection.It is speculated that after BmNPV infection in silkworm cells,the DNA of the gene body regions of these two genes will be hypermethylated,thereby promoting gene expression.The results suggest that DNA methylation may play a role in the interaction between silkworm and BmNPV.In summary,our research has confirmed that DNA methylase can play a role in BmNPV infection.By screening and verifying genes that have significant differences at the transcriptome level and DNA methylation level at the same time,we have preliminarily explored the role of DNA methylation in the interaction between silkworm and BmNPV.The results of this study provide new ideas and research directions for studying the molecular mechanism of the interaction between the silkworm and the virus.