| As the most important modification of epigenetics,DNA methylation plays an important role in many biological processes,such as gene silencing,gene imprinting,X chromosome inactivation,maintenance of chromosome integrity,cell differentiation,development,DNA repair and recombination.A large number of studies have shown that the change of DNA methylation level in promoter region could affect the expression of many immune related genes.In this study,whole genome bisulfite sequencing(WGBS)was used to detect the DNA methylation status of Scophthalmus maximus kidney tissue before injection inactivated Aeromonas salmonicida vaccine(defined as NVSm group)and after injection of inactivated A.salmonicida vaccine(defined as As VSm group),respectively.The differentially methylated regions(DMRs)were compared and the differentially methylated genes(DMGs)were screened.The differential expressed genes(DEGs)of the kidney tissue from NVSm group and As VSm group were screened by RNA-seq.GO and KEGG analysis were used to screen immune related genes.WGBS and RNA-seq results were used to screen differentially expressed immune genes which were regulated by DNA methylation,and then the dual luciferase reporter assay was used to verify the regulatory effect of DNA methylation on gene expression.The results are as follows:1.Changes of whole genome methylation level in turbot kidney after immunization by inactivated vaccineThe average methylation level of As VSm and NVSm groups was 11.06% and11.03% in the whole genome.Among them,the average percentage of methylation C sites in CG sequence environment accounted for 85.67% and 85.63% respectively,indicating that the total DNA methylation level of turbot had no significant change before and after immunization with inactivated vaccine.A total of 8149 DMRs,including 4377 hypermethylated domains(As VSm/NVSm)and 3772 hypomethylated domains(As VSm/NVSm)were screened by differentially methylated regions analysis.GO analysis of DMR related DMGs in CG sequence environment showed that DMGs were mainly enriched into 3105 GO terms,including 1774 GO terms of biological processes,452 GO terms of cell components and 879 GO terms of molecular functions.KEGG enrichment analysis showed that DMGs were significantly enriched in 138 pathways,of which the main immune pathways were MAPK signaling pathway,Wnt signaling pathway and Melanogenesis.Six DMR including C5ar1-Like,EPX-Like,MAP3K15-Like,MAPK14A-Like,MAPK6 and IRF4-Like were screened and sequenced by bisulfite sequencing(BSP).It was found that the methylation status of six DMGs in As VSm and NVSm groups was consistent with that of WGBS,which confirmed the reliability of WGBS results.These results indicated that the DNA methylation level of S.maximus has changed after immunization with inactivated vaccine.This change is not reflected in the overall methylation level,but in the methylation level of individual genes.2.Changes of gene expression level in turbot kidney after immunization by inactivated vaccineTranscriptome sequencing analysis of kidney tissues of As VSm group and NVSm group showed that 386 DEGs with significant difference were screened in As VSm group compared with NVSm group,including 194 up-regulated genes and192 down-regulated genes.GO enrichment analysis of DEGs showed that 147 DEGs were enriched to 249 biological processes,86 DEGs were enriched to 35 cell components,and 214 DEGs were enriched to 166 molecular functions.KEGG enrichment results of DEGs showed that 90 DEGs were enriched in 93 pathways.The immune related pathways included Toll-like receptor signaling pathway,NOD-like receptor signaling pathway,C-type lectin receptor signaling pathway and PPAR signaling pathway pathway.Four immune related DEGs including C5ar1-Like,EPX-Like,MAP3K15-Like and MAPK14A-Like were randomly selected for q RT-PCR to verify the transcriptome sequencing results.The results showed that the expression trend of four immune related DEGs in As VSm group and NVSm group was consistent with that of RNA-seq,which confirmed the reliability of RNA-seq results.The results of RNA-seq showed that the gene transcription levels of several immune related pathways in S.maximus were significantly changed after vaccination with inactivated A.salmonicida vaccine.This means that the inactivated vaccine may activate the innate immunity of S.maximus and regulate the changes of gene transcription by activating immune related pathways,and play a protective role in turbot against A.salmonicida.3.Association analysis of genome-wide methylation level and gene expression levelThrough association analysis of genomic methylation and transcriptome,a total of 45 negative regulatory genes were screened in As VSm group compared with NVSm group,among which 36 negative regulatory genes were located in genebody region,including 8 hypermethylated and low expression genes,28 hypomethylated and high expression genes;9 negative regulatory genes were located in promoter region,including 2 hypermethylated and low expression genes,7 hypermethylated and low expression genes.GO enrichment analysis focused on CG sequence of promoter region showed that there were only five negative regulatory genes(eosinophil peroxidase-like,angiopoietin-related protein 5-like,C5 a anaphylatoxin chemotactic receptor 1-like,arginase-2 mitochondrial,hypothetical protein)were significantly enriched into 74 GO terms,including 41 biological processes,20 cell components and 13 molecular functions.KEGG enrichment analysis showed that the immune related pathways with significant enrichment of negative regulatory genes included MAPK signaling pathway and NOD-like receptor signaling pathway.Two immune related negative regulatory genes(C5a anaphylatoxin chemotactic receptor1-like,C5ar1-Like and eosinophil peroxidase-like,EPX-Like)with DMR located in promoter region were screened for dual luciferase reporter assay.The results confirmed that DNA methylation could affect gene expression by affecting promoter activity.In conclusion,we concluded that the DNA methylation level of C5ar1-Like and EPX-Like gene promoters decreased and their expression level increased after turbot was immunized with inactivated vaccine. |
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