| sea cucumber Apostichopus japonicus has become One of the most important economically species along the northern coastal areas of China.As an environmental factor,temperature has great influence on the A.japonicus' s growth,development and physiological activities.As the global climate change,extreme climate like high temperature in summer occured frequently in many areas of the world.In recent years,the continuous high temperature weather in summer in China has severely affected the pond aquiculture of A.japonicus in Shandong provinces,Liaoning provinces and other regions.A large number of A.japonicus have corrupted and died,causing huge economic losses to the A.japonicus culture industry.At present,with the cultivation scale of A.japonicus expanding increasingly in China,researchers have paid more attention to the growth and the physiological activity of A.japonicus.To research the effects of high temperature stress and the response mechanism of A.japonicus,we can provide an effective strategy for A.japonicus' s cultivation in high temperature,improving quality and survival rate of A.japonicus and optimizing the current breeding mode of A.japonicus,which was of great significance to the health of the aquaculture industry.In this study,the A.japonicus was used as the object of our research,and we uesd RNA-seq technology to analyze the expression rules of differentially expressed genes(DEGs)in the intestine tissue of the A.japonicus after temperature alteration.On this based,to investigate molecular regulation mechanism of A.japonicus' s intestine tissue associated with temperature stress,whole-genome bisulfite sequencing(WGBS)technology was performed to detect differential methylation patterns and analyze the mechanism of differential DNA methylation in A.japonicus coping with temperature stress.Integration of transcriptome data and DNA methylome data could reveal the regulation mechanism of DNA methylation on differentially expressed genes in high temperature stress of A.japonicus.The main results are as follows:1.In order to adequately understand the transcriptome characteristics of A.japonicus' s intestine tissue at different temperatures,we selected 2-year-old A.japonicus under different temperature conditions and constructed a total of 9 c DNA sequencing libraries.After sequencing,the average output of each sample was obtained which was 52,307,882 raw reads.After filtering,there are approximately 7.68 Gb of clean reads.The transcriptome expression profile of the A.japonicus' s intestine tissue was obtained at different temperatures using RNA-seq technology and 17 DEGs were randomly selected and the reliability of sequencing results was verified by quantitative PCR.It was found that the number of DEGs identified by T26 vs T20,T32 vs T20,T32 vs T26 was 59,3479 and 1635,respectively.By KEGG and GO enrichment analysis of DEGs,in the two comparison groups T26 vs T20 and T32 vs T20,most of the DEGs were enriched in the metabolic process,and many DEGs related to immune and epigenetic modification showed significantly up-regulated or down-regulated expression in the T32 vs T20 group.In addition,up-regulation DEGs in the T32 vs T26 group were significantly enriched in the endoplasmic reticulum protein processing pathway,while differentially expressed down-regulated genes were mainly enriched in the peroxisome pathway.It is suggested that these pathways or genes may be involved in the mechanism in response to high temperature 32?.2.Whole-genome methylation analysis.Genome-wide DNA methylation profiles of intestine tissue were constructed at different temperatures.Differentially methylated regions(DMRs)analysis found that in genebody region there were 2199,2254 and 2334 differentially methylated genes(DMGs)in the T26 vs T20 group,T32 vs T20 group and T32 vs T26 group,respectively.In promoter region,there were1015,1022 and 1106 DMGs in the T26 vs T20 group;T32 vs T20 group and T32 vs T26 group,respectively.The enrichment analysis showed that in the T32 vs T20 group DMGs were significantly enriched metabolic process in the promoter region and the genebody region.In the promoter region,DMGs in T26 vs T20 group are mainly enriched in various metabolic processes including carbon metabolism.In genebody,DMGs in the T26 vs T20 group were mainly enriched in peroxisome.In addition,we have also found that DMGs of hypomethylated were enriched in endocytosis,ubiquitin mediated proteolysis and protein processing in the endoplasmic reticulum in the T32 vsT26 group,DMGs of hypermethylated were also enriched in these two pathways in the T26 vs T20 group.It is speculated that these genes may be related to the mechanism of DNA methylation regulation when water temperature from 26 ? to 32 ? in the A.japonicus' s intestine tissue.3.Combined analysis of transcriptome data and genomic methylation data,it was found that there were 3,241 and 108 overlapping genes in genebody region in the T26 vs T20,T32 vs T20 group and T32 vs T26 group,respectively.There were 2,82 and 26 overlapping genes in promoter region in the T26 vs T20,T32 vs T20 group,T32 vs T26 group,respectively.The enrichment analysis was performed on the genes of overlap between DNA methylation and gene expression indicated that in the T26 vs T20group were few genes related to metabolism process.In genebody region,the T32 vs T26group were significantly enriched in the glutathione metabolism related to antioxidant.In T32 vs T20 of genebody region were also significantly enriched in peroxisomes and retinol metabolism related to antioxidant.In addition,it was also found that several genes related to antioxidant showed negative correlation between gene methylation level and expression level in the promoter region or genebody region in T32 vs T20 group.These data indicated that high temperature 32 ? can significantly affect these genes involved in antioxidants,and also indicated that DNA methylation modification in the A.japonicus' s intestine tissue may participate in the molecular mechanism of high temperature adaptation by regulating the methylation status of these genes. |
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