甲基化介导的刺参眠期高温逆境响应机制及胁迫记忆研究

Research on the mechanism of response to high temperature stress of Apostichopus japonicus was urged due to large expansion and severe heat damage in aquaculture. Epigenetic regulation was a new scientific advance in Biological stress response and memory mechanism. In the study, DNA methylation, as an important form of Epigenetic modification, was adopted to investigate stress responses to high temperature of Apostichopus japonicus. Genome methylation characteristics and variation of DNA methylated loci in important functional genes or regions under specific high temperature stress would be detected by the methods of whole genome DNA methylationMeDIP-Seq, bisulfite genomic sequencing PCR, Methylation specific PCR, and so on. Stability and genetic inheritance would also be tracked after the high temperature stress. The study was to reveal the internal regulation of DNA methylation in the process of response to environmental temperature stress of sea cucumber from the perspectives of whole genome level and specific methylation loci, meanwhile, stress memory mechanism was be explored from the aspect of DNA methylation epigenetic modifications. Molecular regulation mechanism of epigenetic modification about high temperature stress response in Apostichopus japonicas, is to be interpreted, which would rich the theory of adaptivity to the changing environment, provide research basis and the scientific basis for breeding and healthy aquaculture of Apostichopus japonicas under the high - temperature environment, and guarantee the healthy and sustainable development of sea cucumber industry in the long run.

刺参养殖规模的扩大化及夏季养殖损失对刺参应答高温胁迫环境的机制提出研究需求。表观遗传修饰调控是生物逆境响应和记忆机制研究的新进展，本项目以DNA甲基化作为表观修饰研究切入点，利用甲基化敏感扩增多态性MSAP、高通量甲基化测序MeDIP-Seq、亚硫酸盐甲基化测序BSP等现代检测手段，扫描和定点检测实验设置高温胁迫下刺参全基因组甲基化特征和差异甲基化区段及功能基因的甲基化位点修饰变化，并跟踪测定胁迫后养殖期及子代甲基化状态，从全基因组水平和特异甲基化位点角度揭示DNA甲基化修饰在刺参应答环境高温胁迫活动过程的内在调控作用，并探析胁迫记忆的基础—胁迫应答DNA甲基化表观遗传修饰的稳定性和遗传继承性。研究旨从表观遗传修饰水平阐释刺参高温胁迫应答的分子调控机制，丰富刺参适应环境变化的理论，为刺参育种和高温环境健康养殖提供研究基础和科学依据，保障刺参产业健康持续发展。

极端性气候如夏季高温频繁地出现，导致刺参养殖业遭受巨大的经济的损失。目前我国刺参养殖规模的日益发展对加强高温胁迫对刺参机体的影响和刺参的应答反应机制提出需求。本研究采用使用全基因组重亚硫酸盐测序（WGBS）技术，构建了仿刺参20℃、26℃、32℃三个代表温度下消化道组织全基因组DNA甲基化图谱，差异甲基化区域分析发现，在基因体区DT26vsDT20组、DT32vsDT20组和DT32vsDT26组分别得到差异甲基化相关基因2199个、2254个和2334个。在启动子区域DT26vsDT20组、DT32vsDT20组和DT32vsDT26组分别得到差异甲基化相关基因1015、1022个和1106个。GO和KEGG富集分析可知DT32vsDT20组，启动子区和基因体区的差异甲基化基因主要富集在化学代谢过程。DT26vsDT20组启动子区DNA差异甲基化基因主要富集在各种代谢过程包括碳代谢，DT26vsDT20组基因体区DNA甲基化差异基因主要富集在过氧化物酶体信号通路。内吞作用，泛素介导的蛋白降解途径以及内质网中蛋白质加工通路等通路在DT32vsDT26组中低甲基化差异基因的KEGG通路中富集程度较高，而在DT26vsDT20组高甲基化差异基因也富集到这两个通路，推测这些基因可能与仿刺参消化道高温处理后的DNA甲基化调控机制有关。同期转录组数据和甲基化数据进行了联合分析，在DT26vsDT20组、DT32vsDT20组和DT32vsDT26组的基因区重叠基因和启动子区重叠基因分别为3个和2个，241个和82个，108个和26个。对DNA甲基化与基因表达的交集基因进行富集分析，T26vsDT20组富集到的基因与细胞内代谢过程有关，DT32vsDT26组基因体区显著富集到抗氧化有关的谷胱甘肽代谢；DT32vsDT20组基因体区，也显著富集到抗氧化相关的过氧化物酶体和视黄醇代谢过程。多个与抗氧化有关的基因在DT32vsDT20组的启动子区或基因体区均体现出基因甲基化水平与表达水平的负相关，仿刺参消化道中DNA甲基化修饰可能通过调控这些基因的甲基化状态参与高温适应分子机制。研究有助于我们了解仿刺参功能性甲基化修饰在环境压力下的响应机制，也为水产养殖动物的表观遗传学应用于优良品种的选育奠定了基础。