刺参Apostichopus japonicus(Selenka)夏眠期间基因表达模式及DNA甲基化基础特征研究

	刺参Apostichopus japonicus(Selenka)夏眠期间基因表达模式及DNA甲基化基础特征研究
	赵业
学位类型	博士
导师	杨红生
	2015-05-18
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生态学
关键词	刺参apostichopus Japonicus(Selenka) 夏眠表达谱 Dna甲基化
摘要	刺参位列“海八珍”之一，具有较高的营养和药用价值，是我国海水养殖产业中单一产值最大的养殖物种。当海水温度升高到一定范围时，刺参会隐藏于较深较安静的海底进入夏眠。在长达4个月的夏眠期间，刺参会表现出明显的休眠特征：停止活动和摄食，消化道萎缩退化，代谢减退及体重明显减轻。夏眠习性无疑大大延长了刺参养殖周期，增加了养殖成本，因此刺参夏眠研究具有重要的科学意义和产业指导意义。目前，关于刺参夏眠的研究主要集中于组织学、生理学、能量及免疫调控等方面，分子生物学层面的研究较少，夏眠的分子调控机理尚不明确。本研究探索分析了刺参夏眠期间的DNA甲基化特征，揭示了刺参夏眠的基因表达全局调控模式，为进一步探讨刺参夏眠分子调控机理提供理论参考。本文以刺参夏眠过程中的三个关键代表性阶段（正常生长期、深度夏眠期和夏眠解除期）的不同组织（肠道、呼吸树、肌肉和体壁）为材料，利用高通量测序等分子生物学相关技术手段，筛选了适合于夏眠期间刺参不同组织的最优内参基因组合；建立了刺参夏眠不同阶段肠道和呼吸树组织表达谱，揭示了刺参夏眠过程中的基因全局表达模式；克隆分析了刺参甲基化修饰系统关键基因Dnmt1和Mbd2/3的cDNA全长及其在夏眠各阶段的表达水平；检测分析了刺参不同组织在夏眠各阶段的基因组DNA甲基化水平变化，初步证实DNA甲基化与刺参夏眠调控密切相关。主要研究结果如下： 1.分析研究了八个常用刺参候选内参基因在夏眠期的表达稳定性，结果表明各候选内参基因在刺参夏眠各组织中呈现差异的表达模式。进一步筛选优化出适合于刺参夏眠期间不同组织的最佳内参基因组合：肠道组织最优内参基因组合为RPS18、TUBB和NADH；呼吸树组织最优内参基因组合为ACTB、TUBB和SDHC；肌肉组织最优内参基因组合为TUBA和NDUFA13。 2.采用RNA-seq技术构建刺参肠道和呼吸树夏眠关键时期的基因表达谱，揭示整个夏眠过程的全局基因表达模式并筛选出一系列参与夏眠调控的关键差异表达基因，分析了肠道和呼吸树组织基因表达的共同性和组织特异性。夏眠期间两种组织差异表达基因主要涉及广泛代谢抑制过程（尤其高耗能生物过程）、能量重调过程以及机体防御与免疫启动过程，而呼吸树组织中细胞周期调控受到明显抑制，表观遗传相关基因转录水平明显变化。这些基因可作为候选基因为后续深入开展夏眠分子调控机理研究搭建平台。 3.克隆获得了刺参DNA甲基化修饰系统中关键基因Dnmt1和Mbd2/3的cDNA全长。刺参Dnmt1基因序列与脊椎动物具有较高的同源性，而Mbd2/3基与脊椎物种的Mbd2和Mbd3基因均具有较高的同源性，且与棘皮动物海胆和海星的Mbd2/3同源关系最近。两者均在刺参深度夏眠期肠道组织中表达显著上调，且mbd2/3在深度夏眠期和夏眠解除期的呼吸树组织中持续显著上调表达，推测上述两基因均参与了刺参夏眠期间的甲基化调控，其作用位点主要在肠道和呼吸树组织。 4.F-MSAP分析表明刺参四种组织基因组DNA均存在甲基化修饰，不同组织甲基化水平分布在28%-56%范围之间。深度夏眠期，肠道和呼吸树中甲基化水平显著升高，且差异主要表现在全甲基化水平上，而半甲基化水平变化并不显著。肌肉和体壁组织在夏眠各阶段的基因组甲基化水平变化不显著。这一结果直接证实了刺参夏眠期间发生了DNA甲基化调控修饰，且调控位点发生于受夏眠影响最为明显的肠道和呼吸树组织中。
其他摘要	Sea cucumber Apostichopus japonicus (Selenka) is of high nutritional and medicinal value, which has the most economic output as a single aquaculture species in China. When sea water temperature rises to some level in summer, A. japonicus will enter into aestivation. The annual period of aestivation usually lasts more than 4 months with obvious torpor characters: complete cessation of feeding and locomotion, loss of body weight, strong metabolism depression and reorganizing of energy. Aestivation undoubtedly shortens the growing cycle of A. japonicus and brings economic losses, hence aestivation research has important scientific significance and industry guidance significance. So far, most studies of sea cucumber aestivation have focused on morphology, physiology and immunology. However, studies of molecular regulatory mechanisms that control aestivation are just beginning. This study explored the DNA methylation characteristics and gene expression profile of A. japonicus during aestivation, which may facilitate the understanding of the molecular regulation mechanisms of aestivation in sea cucumber. In this paper, qRT-PCR, RACE, F-MSAP and high-throughput sequencing techniques were used to analyze the gene expression profile and DNA methylation characteristics of A. japonicus during aestivation. First, we optimized the optimal reference gene combination for different tissues of A. japonicus during aestivation; then we constructed a global gene expression proﬁle of intestine and respiratory tree tissues of A. japonicus using RNA-seq to identify transcriptional responses associated with transitions between different states during aestivation; then we cloned the two key genes Dnmt1 and Mbd2/3 of the DNA methylation system in A. japonicus and analyzed the expression levels during aestivation; at last, we analyzed DNA methylation levels of various tissues of A. japonicus during key aestivation states. Main results were as follows: 1. The expression stability analysis of eight candidate reference genes in sea cucumber showed the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), β-tubulin (TUBB) and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB) TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). 2. We have then analyzed the global gene expression proﬁle of intestine and respiratory tree tissue of A. japonicus during aestivation by constructing and screening three libraries representing key stages of aestivation: non-aestivation(NA), deep-aestivation (DA), and arousal from aestivation (AA) using RNA-seq. A set of respiratory tree speciﬁc DEGs was identiﬁed the ﬁrst time and common DEGs that were responsive to aestivation in both respiratory tree and intestine were then identiﬁed involving with metabolism, immune defense and energy and cell cycle regulation. Most key DEGs in both sea cucumber tissues during aestivation were associated with metabolic depression, especially energy-expensive processes, detoxiﬁcation and tissue protection, and metabolism and energetic reorganization, which may be excellent candidates for further investigating molecular mechanism of aestivation in sea cucumber. 3. Two key genes Dnmt1 and Mbd2/3, which comprise the DNA methylation system in A. japonicus were cloned and analyzed. The deduced amino acid sequences and characteristic motifs of sea cucumber Dnmt1 and Mbd2/3 showed high homology to those of their mammalian counterparts. Quantitative real-time RT-PCR analysis showed Dnmt1 expression in the intestine was up-regulated during deep-aestivation (P<0.05), while Mbd2/3 was over-expressed in both the intestine and respiratory tree during the same period (P<0.01). The results of this study suggest that the above genes may be involved in DNA methylation of sea cucumber during aestivation, and the major sites are the intestine and respiratory tree. 4. DNA methylation levels of A. japonicus were further analyzed by the ﬂuorescence-labeled methylation-sensitive ampliﬁed polymorphism (F-MSAP) technique over three stages of the aestivation cycle. The incidence of DNA methylation was different across tissue types in the non-aestivation stage ranging from 28% to 56%. Our results show that hypermethylation accompanied deep-aestivation in intestine and respiratory tree tissues of A. japonicus, and that full-methylation but not hemi-methylation levels exhibited signiﬁcant increases in the deep-aestivation stage. No significant differences were observed in DNA methylation levels of muscle and body wall tissues over the three aestivation states. Hence we speculate that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation and the main DNA modiﬁcation sites were focused on intestine and respiratory tree tissues, which were most affected organs during aestivation.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/23248
专题	海洋生态与环境科学重点实验室
推荐引用方式 GB/T 7714	赵业. 刺参Apostichopus japonicus(Selenka)夏眠期间基因表达模式及DNA甲基化基础特征研究[D]. 北京. 中国科学院大学,2015.

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