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美洲牡蛎 (Crassostrea virginica) 抗逆转录调控机制及牡蛎进化分析
其他题名Transcriptome Regulation of Stress Response in the Eastern Oyster (Crassostrea virginica) and Oysters’ Phylogenetic Analyses
李翠1,2
学位类型博士
导师郭希明
2017-05-25
学位授予单位中国科学院大学
学位授予地点北京
学位专业理学博士
关键词美洲牡蛎 转录组 环境胁迫 候选基因重测序 Snp 系统发育分析
摘要  最早有化石记录的牡蛎发现于2.5亿年前,此后,经历了多次大灭绝,在白垩纪开始繁盛,现在仍旧有很多种类。牡蛎的多样性以及对极端环境和多变环境的适应性研究刚刚开始。牡蛎基因组项目的实施和完成,帮助我们从分子角度理解牡蛎对潮间带的适应性,揭示了长牡蛎适应环境的关键因素。作为它的姐妹种,美洲牡蛎 Crassostrea virginica,具有更强的抗逆性,但是相关研究较少。本次研究对逆境环境的美洲牡蛎进行转录组测序和抗逆候选基因重测序,鉴定环境胁迫相关的基因,提出转录组水平美洲牡蛎抗逆调控模式解释模型。初步揭示了美洲牡蛎分别在抗高温,低温,干露,低盐等恶劣环境下的转录响应机制及随时间的变化。主要结论如下:
1.牡蛎的长时程抗逆过程分为警报期,抵抗期,衰竭期三个阶段,但是牡蛎的个体水平衰竭期却是转录水平最活跃的时期。相同的胁迫环境下,各阶段差异表达的基因种类不同,相同的基因在不同的阶段的表达量也有变化。美洲牡蛎的热激蛋白、凋亡、免疫、代谢和内分泌系统对牡蛎适应潮间带环境至关重要。
与干露胁迫和低盐胁迫相比,高温胁迫上调的基因最多。随胁迫时间延长,应激相关的基因数目逐渐增多,表达量也增高。但在胁迫第三天时出现相对表达量下降,基因表达下调的数目增加。此时高温胁迫导致的过度能量消耗和细胞损伤可能会增强内质网应激并激活凋亡信号通路 。
牡蛎具有很强低渗胁迫调节能力,通常牡蛎通过调节自由氨基酸含量进行渗透压调控。本次试验设置了处于牡蛎耐受临界点的低盐胁迫,此时美洲牡蛎通过溶质转运蛋白、细胞外基质重塑对渗透压进行调节。干露胁迫时牡蛎出现大量的核糖体蛋白高表达,氨基酸和脂质代谢可能是主要的能量来源。 低温胁迫转录组分析表明:牡蛎适应低温的能力主要来自对生命活动的抑制作用。牡蛎有机体出现衰竭时,濒临死亡的牡蛎转录组出人意料的表达活跃。高温和干露组的蛋白质稳定相关基因和抑制凋亡相关基因均增强表达。
2. 基于Ampliseq方法对1002个候选的抗性关键基因进行重测序,证实在逆境环境下存活与死亡的牡蛎野生群体的等位基因频率发生改变。这些筛选到的大量基因及其SNP位点,可能与牡蛎抗环境压力相关。
3. 我们采集了缅甸和秘鲁的牡蛎样本,进行系统发育分析,发现秘鲁有 Undulostrea megodon Hanley, 1846 和 Talonostrea talonata Li & Qi, 1994 的分布,并根据系统发育分析结果为两种牡蛎分别订名为 Ostrea megodon Hanley, 1846 和 Crassostrea talonata Li & Qi, 1994。我们在缅甸发现三种牡蛎。 Saccostrea malabonensis Faustino 1932 和 Crassostrea gryphoides tanintharyiensis为首次报道。并获得了 Crassostrea belcheri Sowerby 1871在缅甸存在的分子证明。这些发现强调了对牡蛎用分子工具进行分类的重要性,对于我们理解牡蛎的全球多样性、物种分布、适应性以及人类活动引起的物种迁移具有重要意义。
其他摘要Oysters first appeared in fossil records about 250 million years ago. Since then, they have experienced several mass extinctions. Oysters flourished during the cretaceous period, with many living species remaining today. Oyster diversity and their adaptation to challenging intertidal environments are not well understood. The sequencing of the oyster genome has provided opportunities to study the molecular mechanisms of oyster’s adaptation. Studies have revealed key elements of Pacific oyster’s adaptation. Its sister-species, eastern oyster Crassostrea virginica, that shows stronger resistance to environmental stress has received little attention. In this study, we sequenced transcriptomes of eastern oysters under different stresses and resequenced candidate genes to under eastern oyster’s remarkable resilience and identify candidate stress resistance genes. The following are the main findings:
1. Transcriptome response under environment stress
Oyster’s response to environment stress conforms to three different stages: alert, resistance and exhaustion. Transcriptome response differs among different stages. Genes and pathways related to apoptosis, immunity, metabolism and endocrine system are important to oyster’s stress response.
Compared with air exposure and low salinity stress, high-temperature induced upregulation of the largest number of genes. The number of differentially expressed genes increased over time, but declined on the third day. Excessive energy consumption and cellular damage may be the leading causes of death.  Endoplasmic reticulum stress response and apoptosis pathways play important roles in heat resistance.
Eastern oyster has remarkable tolerance to low salinity and air exposure. Its response to low salinity stress involves channel proteins and free amino acid pool as well as the lipid metabolism. A large number of ribosomal proteins are up-regulated under air exposure. Under cold stress, oysters turn to anaerobic respiration to reduce energy supply and other metabolic activities.  Dying oysters show unexpectedly high activities of gene transcription, with genes related to translation and apoptosis inhibition being most active in both heat and air exposure treatment groups.
2. Resequencing of 1002 candidate stress-resistance genes before and after stress-caused mortalities using AmpliSeq identified a set of genes whose allele frequency showed significant changes. These genes and variations may be important to the eastern oyster’s remarkable resistance to environmental stress.
3. We identified two Ostreid oysters from Peru as Undulostrea megodon Hanley, 1846 and Talonostrea talonata Li & Qi, 1994, and renamed U. megodon as Ostrea megodon Hanley, 1846 and T. talonata as Crassostrea talonata Li & Qi, 1994. We identified three oyster species from Myanmar: Crassostrea belcheri Sowerby 1871, a common species found in Indo-Pacific; Saccostrea malabonensis Faustino 1932 and Crassostrea gryphoides tanintharyiensis. They are the first confirmed records in Myanmar. These findings highlight the need for molecular classification of oysters to understand oyster’s global diversity, distribution, adaptation and human-facilitated transfers. 
学科领域海洋生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136623
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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李翠. 美洲牡蛎 (Crassostrea virginica) 抗逆转录调控机制及牡蛎进化分析[D]. 北京. 中国科学院大学,2017.
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