| Sea cucumber(Apostichopus japonicus)belongs to Echinoderms,it has important economic value and nutritional value and is an important aquaculture species in coastal areas such as Hebei,Dalian,Shandong,and Fujian in China.In recent years,the culture industry has expanded dramatically,many problems such as disease and large-scale death caused by extra high-water temperature in summer have led to serious economic loss for the culture.Therefore,new varieties with disease resistance and high temperature resistance are urged in the culture industry.And the corresponding research on molecular mechanism of the disease resistance and high temperature tolerance of A.japonicus will provide basis for the selective breeding.In this study,whole genome bisulfite sequencing(WGBS)and transcriptome sequencing using high-throughput sequencing technology were conducted to reveal the changes of DNA methylation and gene expression in the body wall of A.japonicus under high temperature and Vibrio splendidus infection stress,and the prediction of differential lnc RNA target genes were selected,then key loci and associated genes were isolated by conjoint analysis.The results are listed as follows:1.Different methylation analysis of the body wall of A.japonicus under high temperature and pathogen infection stressThe body wall tissues of A.japonicus were obtained by artificial infection challenge and high temperature stress,and the WGBS genomic DNA methylation sequencing of the body wall tissues was performed using the unchallenged group and healthy body wall tissues at 13℃ as the control group to investigate the genomic DNA methylation level of A.japonicus body wall and screen the differentially methylated regions and related genes of the corresponding pathogenic stresses to provide basic data for dissecting A.japonicus response temperature and pathogenic stresses from epigenomics.The results of methylation analysis showed that the total methylation levels of A.japonicus genome were(3.59±0.04)% and(3.87±0.27)% in the control and infection groups,respectively,and(3.70±0.03)% and(3.94±0.17)% in the control and high temperature groups,respectively,and the methylation levels of A.japonicus genomic DNA were significantly increased under pathogenic and high temperature stresses(P < 0.05),and the mean methylation levels of Cp G,CHG,and CHH were increased under pathogenic stresses,while the mean methylation levels of Cp G and CHH were increased under high temperature stresses.Among the methylated sites,m Cp G was the most important methylation form.In the pathogen infection challenge group 626,677 DMRs were screened,of which 333,976 DMRs were in higher methylation levels and 292,701 DMRs were in lower methylation levels,23,706 genes were annotated for the DMRs.In the high temperature challenge experimental group,748,347 DMRs were screened,of which 495,122 DMRs were in higher methylation levels and 253,225 DMRs were in lower methylation levels,and 25,387 genes annotated.Both the number of DMRs with increased methylation levels was higher than the number of DMRs with decreased methylation levels under pathogen and high temperature.GO enrichment analysis of gene annotation of DMRs regions showed that differentially methylated genes were mainly concentrated in terms related to negative regulation of transcription by RNA polymerase II,Golgi membrane and protein binding function;KEGG functional annotation showed that pathogen and high temperature mainly affected MAPK signaling pathway,RNA transport pathway and endocytosis pathway.Protein tyrosine kinase,ubiquitin-conjugating enzyme,ribosomal protein,Hsp70 and other genes are differentially methylated genes under pathogenic and high temperature groups,while THAP domain and integrin plexin domain are unique differentially methylated genes under pathogenic challenge,and Ran BP1 and lipase are unique differentially methylated genes under high temperature challenge.The result would give foundation for further studies on the mechanism of DNA methylation on the regulation of pathogenic infection and high temperature stress.2.Different gene expression of the body wall of A.japonicus under high temperature and pathogen infection stressBioinformatic analysis showed that a total of 29,290 genes were detected by transcriptome sequencing,496 DEGs were screened in the pathogenic challenge experimental group,of which 214 were up-regulated and 282 were down-regulated,and1,409 DEGs were screened in the high temperature challenge experimental group,of which 881 were up-regulated and 528 were down-regulated.KEGG gene annotation of DEGs showed that 68 and 170 significant differential genes were enriched in 20 pathways in the pathogenic and high temperature experimental groups,respectively,and all of them were the most genes in protein processing pathways in the ER,and pathogenic infection and high temperature were mainly related to transmembrane proteins and Hsp70,respectively.Among the cis-regulated significantly different lnc RNAs and m RNAs in the pathogenic stress experimental group,interferon-induced tetrapeptide repeat protein 5 has a high possibility of regulatory relationship.There were40 cis-regulated significant differential lnc RNA target genes and 26 differentially regulated m RNAs in the heat stress experimental group,and they were highly correlated.GO enrichment analysis of lnc RNA-targeted differential genes in the pathogen and high temperature experimental groups showed that the three annotated genes of biological process,cellular component and molecular function ontology were mainly related to biological process,cytoplasm,and metal ion binding.KEGG enrichment analysis showed that pathogenic stress mainly affected phagosomes pathways,while high temperature stress mainly affected longevity regulating pathway-multiple species,and the relevant results provide basic data for transcriptome-level dissection of A.japonicus response to temperature and pathogenic stress mechanisms.3.Conjoint analysis of genome-wide DNA methylation and transcriptome sequencingNegative correlation genes were screened by combined genomic methylation and transcriptome analysis,and 180 negative correlation genes were screened in the pathogenic stress experimental group,of which 60 negative correlation genes with differentially methylated regions located in promoter regions;GO and KEGG enrichment analysis of negative correlation genes screened related pathways and key genes such as LRR,Hsp20 and CARD.In the high temperature stress experimental group,569 negatively correlated association genes were screened,of which 223 negatively correlated genes with differentially methylated regions located in promoter regions;GO and KEGG enrichment analysis of negatively correlated association genes screened key genes such as related pathways and adenylyl cyclase.Eight negatively correlated genes were verified by bisulfite sequencing PCR(BSP)and quantitative real-time PCR(q RT-PCR).The result showed that the methylation level of transcription factor E1 K changed in response to pathogenic stress and affected the expression of this gene,and the methylation level of three genes,U11/U12 micronucleated ribonucleoprotein 35 k Da,BSL78＿12691,and Hsp70,changed in response to temperature stress and affected the expression of this gene.The corresponding results provide basic data for dissecting the mechanism of epigenetic differences on transcriptional expression in A.japonicus in response to pathogens and temperature stress. |
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