刺参基因组DNA甲基化水平及模式对温度变化的响应

DNA methylation level of genomic DNA of Apostichopus japonicus at different temperatures

中文关键词: 刺参  DNA甲基化水平及模式  休眠  高温胁迫  全基因组重亚硫酸盐测序技术  酶联免疫吸附

英文关键词: Apostichopus japonicus  DNA methylation and mode  Dormancy  High temperature stress  Whole genome bisulfite sequencing  Enzyme linked immunosorbent assay

中文摘要:

      本研究为了探讨刺参(Apostichopus japonicus)在不同温度胁迫下的DNA甲基化水平与甲基化模式变化，利用全基因组重亚硫酸盐测序技术(WGBS)和酶联免疫吸附法(ELISA)对刺参在3个温度下(20℃、26℃和32℃)的纵肌、呼吸树、消化道和体壁4个组织进行分析。WGBS测序结果显示，在消化道组织中，20℃、26℃和32℃温度组全基因组总甲基化水平分别为(1.70±0.01)%、(1.79±0.11)%和(1.59±0.04)%，26℃组处于休眠状态，刺参消化道基因组甲基化水平升高，而32℃组高温胁迫下，甲基化水平下降；在总甲基化位点中，CG类型是主要的甲基化修饰(96%以上)，CHH和CHG位点占比较低；30%甲基化水平的甲基化位点中，CHG和CHH为本类型甲基化的最高点，且显著高于CG类型。ELISA检测结果显示，3种不同温度下，刺参呼吸树和消化道组织的甲基化水平范围为2.68%~3.29%，均高于纵肌和体壁组织；温度变化后，刺参呼吸树和消化道组织的总甲基化水平均有显著变化，而纵肌和体壁的总甲基化水平基本不变，表明DNA甲基化可能参与刺参的高温胁迫调控机制。刺参应对温度变化过程中DNA甲基化水平的研究，从表观遗传学视角解析温度升高对刺参不同组织的影响，可为丰富刺参甲基化研究内容和无脊椎动物的甲基化发生规律提供参考。

英文摘要:

      The sea cucumber Apostichopus japonicus requires its own defense mechanism to resist and adapt to high temperature stress. Epigenetic modification plays an important role in this regulation process. To investigate the changes of DNA methylation level and methylation pattern in A. japonicus (2-year-old) under different temperature stress, whole genomic bisulfite sequencing (WGBS) and enzyme-linked immunosorbent assay (ELISA) were used to detect whole genome methylation levels of the muscle, respiratory tree, digestive tract, and body wall. Three temperature gradients of 20℃, 26℃, and 32℃ were set up in this experiment. The results of WGBS showed that the total genome wide methylation levels of 20℃, 26℃, and 32℃ groups were (1.70±0.01)%, (1.79±0.11)%, and (1.59±0.04)%, respectively. The methylation level of the digestive tract genome of A. japonicus in the 26℃ group increased, while the methylation level of the 32℃ group decreased under high temperature stress. Among the total methylation sites, CG type was the main site of methylation modification (more than 96%), and CHH and CHG sites showed relatively low levels of modification. Among the methylation sites at 30% methylation level, CHG and CHH methylation sites showed the highest methylation, and were significantly higher than that of the CG type. The results of ELISA showed that the methylation levels of respiratory tree and digestive tract tissue ranged from 2.68% to 3.29% at three different temperatures, which were higher than those in muscle and body wall tissue. After temperature change, the total methylation level of respiratory tree and digestive tract tissue of sea cucumber changed significantly, while the total methylation level of muscle and body wall remained unchanged, indicating that DNA methylation may be involved in the regulation mechanism of high temperature stress in A. japonicus. The study on DNA methylation level of A. japonicus in response to temperature change can be used to analyze the effect of temperature rise on different tissues of sea cucumber from the perspective of epigenetics. This research can provide reliable basis for enriching the research content on methylation in A. japonicus and the occurrence of methylation in invertebrates.