实验海洋生物学重点实验室知识库

全球变暖背景下，海洋生物的生存和繁殖等受到严重威胁。栖息于环境波动剧烈的潮间带区域的牡蛎面临昼夜和季节性的剧烈环境波动，其广泛的分布为研究温度适应性与进化提供良好模型。此外，牡蛎不仅是世界性的经济养殖贝类和我国贝类养殖的传统支柱产业，还为海洋生态系统提供多种多样的生态服务功能。近年来，由于海水温度升高以及骤变频率增强等原因，牡蛎的夏季大规模死亡现象频发为全球牡蛎产业带来巨大经济损失。气候变化和人为活动的破坏还加剧了世界野生牡蛎资源及牡蛎礁的退化。因此深入探究牡蛎温度适应性调控机制不仅具有极高的科学价值，还具有重要的生态和产业意义。各种适应性以及农艺性状的形成是自然选择下生物达到环境最佳适合度的结果，因此研究适应性性状关键基因及其调控是深入理解适应性进化机制的核心。本研究选取异域分布的差异温度适应性的近缘种—长牡蛎（Crassostrea gigas）和福建牡蛎（Crassostrea angulata）为研究对象，通过同质化养殖和对调养殖，表型测量和多组学联合分析从基因组差异-基因表达调控-蛋白-蛋白翻译后修饰等多层级水平鉴定介导二者温度适应性性状—脂肪酸和耐高温分化的关键基因，并通过分子功能实验验证相关基因功能，挖掘关键调控元件及解析上游信号转导通路。相关研究结果有助于理解海洋生物温度适应性分化和进化背后的遗传和分子机制，为全球变暖背景下预测海洋生物的适应性潜力提供帮助，并为牡蛎品质和抗性性状协同改良提供理论基础和育种元件。
1、脂肪酸性状分化关键基因及其调控机制
利用在山东青岛（35° 44′ N）进行同质化养殖的长牡蛎和福建牡蛎进行表型测定和转录组代谢组联合分析，发现长牡蛎和福建牡蛎之间在脂肪酸性状上存在显著分化，特别是不饱和脂肪酸—油酸（C18：1）含量在长牡蛎中显著高于福建牡蛎。此外，福建牡蛎表现出更强的生长性能和有氧代谢能力，而长牡蛎表现出更高的糖原含量和抗氧化能力。转录组与代谢组联合分析结果显示长牡蛎抑制糖酵解和脂肪酸氧化通路，上调脂肪酸合成和抗氧化相关基因和代谢物，进而将能量更多地分配给储存和防御。而福建牡蛎上调蛋白质合成基因和代谢物，并具有更高的生长相关生物标志物含量，进而将更多能量分配给生长。研究结果揭示了介导两种牡蛎生长-防御权衡（Trade-Offs）的分子机制，为适应性性状的协调性平衡及其背后的生化和分子基础提供新见解。
基于上述实验结果以及团队前期的两物种的群体基因组比较研究，本研究锁定催化合成单不饱和脂肪酸——油酸的关键限速酶硬脂酰辅酶A去饱和酶（Stearoyl-CoA Desaturase，Scd）作为介导长牡蛎和福建牡蛎脂肪酸性状分化的关键基因。基因功能实验证实牡蛎SCD基因可以催化合成单不饱和脂肪酸棕榈酸（C16:1）和油酸（C18:1），并增强细胞膜流动性。启动子区遗传变异位点筛查和功能验证显示牡蛎Scd启动子区的变异通过创造（长牡蛎）或破坏（福建牡蛎）正向转录因子Y Box Factor的结合基序来增强长牡蛎Scd基因的基础表达和油酸基础含量。而受低温诱导的正向转录因子Srebp通过其差异低温响应模式（福建牡蛎上调，长牡蛎下调）参与塑造福建牡蛎更高的Scd基因塑性表达（上调幅度），Scd基础表达和塑性表达在两种牡蛎间存在权衡（Trade-Offs）。相关研究结果表明脂肪酸性状，特别是不饱和脂肪酸，既是一种重要的能量物质，也是重要的细胞结构物质，其参与介导生物的温度适应。长期的差异环境温度塑造了长牡蛎和福建牡蛎分化的不饱和脂肪酸及其代谢基因可塑性模式，即性状均值和可塑性之间的权衡，顺式（Cis-）和反式（Tans-）变异共同介导牡蛎Scd基因可塑性的分化和进化模式。本研究作为一个研究案例揭示了环境响应性性状普遍存在的差异表型可塑性分化模式背后的遗传和分子机制，进一步加深对表型可塑性进化机制的理解，并为适应性性状的形成和海洋生物面对未来气候变化适应潜力的预测提供新见解。随后，利用长牡蛎和福建牡蛎杂交F2群体进行油酸含量和遗传变异的关联分析，结果进一步证实上述筛选到的遗传变异与表型显著相关，且优势基因型组合（长牡蛎）个体油酸含量显著高于劣势基因型组合（福建牡蛎）个体，相关等位基因变异组合可用于基于分子模块辅助选育的牡蛎脂肪酸性状遗传改良。
2、耐高温性状分化关键基因及其调控机制
热胁迫状态下的长牡蛎和福建牡蛎的表型评估发现福建牡蛎具有更高的有氧代谢能力和抗氧化能力，以及更低的组织凋亡率，研究结果进一步支持福建牡蛎具有更强的耐热性。后续利用染色质动力学，基因表达，蛋白组和磷酸化蛋白组等多组学手段对热胁迫下的长牡蛎和福建牡蛎进行比较研究以鉴定二者差异热耐受的关键基因和相关信号通路。RNA-Seq和ATAC-Seq数据显示凋亡抑制蛋白（IAP）家族及其相关的细胞凋亡途径是介导二者耐热性分化的主要信号通路。长牡蛎可能主要通过转录因子（如Forkhead Box Family）激活分子伴侣基因，特别是热休克蛋白以响应高温。而福建牡蛎则激活抗凋亡、DNA损伤修复和脂肪酸合成相关基因。蛋白组和磷酸化蛋白组数据显示高温通过影响蛋白含量和磷酸化水平来调控信号传导、能量代谢、蛋白质合成、细胞存活和凋亡以及细胞骨架重塑等生物学过程，差异磷酸化修饰的蛋白激酶A（PKA）、丝裂原活化蛋白激酶1（ERK1）、酪氨酸蛋白激酶Src（SRC）和丝氨酸/苏氨酸激酶AKT可能是潜在的枢纽调控基因（Hub Regulatory Gene），其通过增强糖酵解和三羧酸循环以增加能量供给，调控蛋白质合成，抑制内源线粒体细胞色素C释放引发的Caspase依赖性凋亡以及维持细胞骨架稳定性来介导福建牡蛎的高耐热性。本研究结果首次从基因表达调控-蛋白-蛋白翻译后修饰等多层级分子调控水平对长牡蛎和福建牡蛎差异热响应的细胞动态变化进行全面评估，并挖掘了大量介导二者热抗性分化的关键基因及其磷酸化位点。
后续研究中，我们选择糖酵解关键限速酶—丙酮酸激酶（PK），NF-κB的核心调控因子—NF-κB的抑制蛋白（IκBα）和细胞凋亡关键执行者—半胱氨酸天冬氨酸蛋白酶3（Caspase3）进行后续磷酸化功能及上游调控通路研究。
①长牡蛎和福建牡蛎IκBα Ser74位点的磷酸化水平在热胁迫前后存在显著差异，即福建牡蛎上调，长牡蛎下调。磷酸化功能实验发现该位点能够独立介导其泛素-蛋白酶体降解并降低热稳定性，这与模式生物中已建立的IKKs激酶磷酸化IκBα Ser32和Ser36位点进而控制其蛋白降解的机制不同。该位点在牡蛎主效IκBα中特异性进化，且被ERK1/2激酶磷酸化，并进一步受到经典MAPK通路调节。牡蛎IκBα在热胁迫下的差异磷酸化可促进福建牡蛎更强的REL1和REL2热响应入核，激活细胞存活、脂肪酸代谢、蛋白翻译和与抗氧化相关基因的表达，从而帮助其抵御热胁迫。研究结果首次报道了后生动物中保守的免疫明星通路—NF-κB的核心调控因子IκBα在牡蛎中存在一个全新的热诱导磷酸化位点（Ser74）。牡蛎高温-RTK-MRAS-BRAF-MAPK-NF-κB信号级联在长牡蛎和福建牡蛎之间存在差异的热响应和温度适应模式，表明其参与塑造二者差异温度适应和热存活。
②长牡蛎和福建牡蛎Caspase-3/7 Thr260位点的磷酸化水平在热胁迫前后存在显著差异，即福建牡蛎上调，长牡蛎下调。基于系统进化分析和序列比对发现双壳纲中存在一组特异性进化的具有长亚基间连接区（IL）的新型Caspase-3/7基因，进一步的体内和体外功能实验发现长IL可以抑制双壳纲特异性CASP3/7裂解激活。位于IL中的Thr260位点在双壳纲中保守，并被AKT激酶磷酸化，进而抑制CASP3/7的热诱导激活。Thr260位点磷酸化受到经典PI3K-AKT通路调节，并在长牡蛎和福建牡蛎之间呈现出分化的高温响应模式，表明双壳纲特异性PI3K-AKT-CASP3/7信号级联参与介导二者的差异热诱导凋亡。研究结果首次在非模式生物中报道PI3K-AKT-CASP磷酸化调控通路，为软体动物中复杂且独特的凋亡机制提供新见解。
③长牡蛎和福建牡蛎PK Ser11位点的磷酸化水平在热胁迫前后存在显著差异，即福建牡蛎上调幅度高于长牡蛎。Ser11位点在双壳纲和腹足纲中保守，该位点磷酸化可以通过增强底物结合来提升PK酶活，进而促进糖酵解以提升ATP合成来帮助牡蛎热损伤修复。进一步针对上游调控通路的功能验证发现ERK1/2激酶磷酸化PK Ser11位点，其同样受到经典MAPK通路调节，软体动物（双壳纲和腹足纲）特异性高温-RTK-MRAS-BRAF-MAPK-PK信号级联在长牡蛎和福建牡蛎间存在差异的热响应和温度适应模式，表明磷酸化通过调控糖酵解速率来影响二者热胁迫下差异的能量代谢能力进而介导分化的耐热性。
上述研究结果凸显了传统保守通路在海洋生物中存在复杂而独特的磷酸化介导的调控网络，其通过影响细胞存活、细胞凋亡和能量代谢来介导异域分布的近缘种长牡蛎（相对高温敏感种）和福建牡蛎（相对高温耐受种）间差异温度适应和耐热性，研究结果进一步拓展我们对现有经典通路之间的信号串扰（Cross-talk）机制在进化和功能方面的理解。此外，研究中筛选到的热抗性关键效应基因和上游调控因子及其对应磷酸化位点为牡蛎耐热性性状遗传改良提供理论基础和重要靶点。

In the context of global warming, the survival and reproduction of marine organisms face significant threats. Oysters inhabiting the highly fluctuating intertidal zone experience drastic environmental fluctuations on a diurnal and seasonal basis, and its wide distribution making them an excellent model for analyzing temperature adaptation and evolution. In addition, oysters are not only globally recognized as economically important cultured mollusks and a traditional pillar industry in China's shellfish aquaculture, but also provide various ecological services to marine ecosystems.However, in recent years, the occurrence of large-scale mortality events during summer due to increased seawater temperatures and intensified temperature fluctuations has caused substantial economic losses in the global oyster industry.  Climate change and human activities have also exacerbated the degradation of wild oyster resources and oyster reefs worldwide. Therefore, investigating the mechanisms underlying oyster temperature adaptation is not only of great scientific value but also holds significant ecological and industrial implications. The formation of various adaptive and agronomic traits is the result of natural selection driving organisms to achieve optimal fitness in their environment. Therefore, studying key genes and their regulation involved in adaptive traits is crucial for a deeper understanding of the mechanisms underlying adaptive evolution. In this study, we focused on two congeneric oyster species with divergent temperature adaptation and allopatric distribution, Crassostrea gigas (C. gigas) and Crassostrea angulata (C. angulata). By employing common garden and reciprocal transplant experiments, combined with phenotypic measurements and multi-omics analyses spanning genomic variations, gene expression regulation, protein abundance, and post-translational modifications, we aimed to identify the key genes mediating the divergence of temperature adaptive traits (fatty acids and heat tolerance). Furthermore, we conducted molecular functional experiments to validate the functions of relevant genes, explore key regulatory elements, and unravel upstream signaling transduction pathways.Our findings will contribute to a better understanding of the genetic and molecular mechanisms underlying temperature adaptation and evolution in marine organisms, assist in predicting the adaptive potential of marine organisms in the context of global warming and provide theoretical foundations and breeding elements for synergistic improvement of oyster quality and resistance traits.
1. Key Genes and their Regulatory Mechanisms underlying Fatty Acid Traits Divergence
Phenotypic measurements and transcriptomic-metabolomic combined analysis were conducted on C. gigas and C. angulata after one-generation of common garden experiments in Qingdao, Shandong Province (35° 44′ N). The results showed a significant differentiation in fatty acid traits between C. gigas and C. angulata, particularly in the content of unsaturated fatty acid—oleic acid (C18:1), which was significantly higher in C. gigas. Additionally, C. angulata exhibited stronger growth performance and aerobic metabolic capacity, while C. gigas showed higher glycogen content and antioxidant capacity. The combined analysis of transcriptomics and metabolomics revealed that C. gigas suppressed glycolysis and fatty acid oxidation pathways, upregulated fatty acid synthesis and antioxidant-related genes and metabolites, thereby allocating more energy to storage and defense. While, C. angulata upregulated protein synthesis related genes and metabolites, with higher levels of growth-related biomarkers, allocating more energy to growth. This study elucidated the molecular mechanisms mediating the growth-defense trade-offs between these two oyster species, providing new insights into the coordinated balance of adaptive traits and their biochemical and molecular basis.
Based on above experimental results and previous genomic comparisons of these two species, this study foucused on Stearoyl-CoA Desaturase (Scd), a key rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acids, particularly oleic acid, as the important gene mediating the fatty acid trait divergence between C. gigas and C. angulata. Functional verification of the Scd gene demonstrated its ability to catalyze the synthesis of monounsaturated fatty acids palmitoleic acid (C16:1) and oleic acid (C18:1) and enhance cell membrane fluidity. Screening and functional validation of genetic variation in the promoter region revealed that variations in the Scd promoter region enhanced the basal expression of the Scd gene and oleic acid content of C. gigas by creating (C. gigas) or disrupting (C. angulata) the binding motif of the positive transcription factor Y Box Factor. Additionally, the positive transcription factor Srebp, through its differential low-temperature response pattern (upregulation in C. angulata, downregulation in C. gigas), was involved in shaping the higher plastic expression (upregulation amplitude) of the Scd gene in C. angulata. The basal expression and plastic expression of Scd in these two oyster species exhibited trade-offs. Our results indicate that fatty acid traits, particularly unsaturated fatty acids, play important roles as energy substances and cell structural materials in mediating temperature adaptation in organisms. Long-term differential environmental temperatures have shaped the divergent plasticity patterns of unsaturated fatty acids and their metabolic genes in C. gigas and C. angulata, involving a trade-off between trait mean and plasticity. Both cis- and trans-variations jointly mediate the plasticity divergence and evolution patterns of the Scd gene in oysters. This research, as a study case, reveals the genetic and molecular mechanisms behind the common phenotypic plasticity differentiation patterns of environmentally responsive traits, further deepening the understanding of the evolutionary mechanisms of phenotypic plasticity and providing new insights into the formation of adaptive traits and the prediction of potential of marine organisms to future climate change. Subsequently, association analysis of oil content and genetic variation was conducted using a hybrid F2 population of C. gigas and C. angulata. The results further confirmed that above screened genetic variations were significantly correlated with phenotype, with individuals of the dominant genotype combination (C. gigas) having significantly higher oil content than those of the inferior genotype combination (C. angulata). These allelic variations combinations can be used for molecular module-assisted selection breeding of oyster fatty acid trait genetic improvement.
2. Key Genes and their Regulatory Mechanisms underlying Heat Tolerance Traits Divergence
Phenotypic assessments of C. gigas and C. angulata under heat stress conditions revealed that C. angulata exhibited higher aerobic metabolic capacity, antioxidant capacity, and lower tissue apoptosis rates, further supporting its stronger heat tolerance. Subsequent comparative studies using chromatin dynamics, gene expression, proteomics, and phosphoproteomics among C. gigas and C. angulata under heat stress aimed to identify key genes and related signaling pathways responsible for their divergent heat tolerance. RNA-Seq and ATAC-Seq data indicated that the Inhibitor of Apoptosis Protein (IAP) family and its associated apoptosis pathway were the main signaling pathway mediating the differentiation in heat tolerance between these two species. C. gigas activated molecular chaperone genes, particularly heat shock proteins, through transcription factors such as Forkhead Box Family, in response to high temperatures. While, C. angulata activated genes related to anti-apoptosis, DNA damage repair, and fatty acid synthesis. Proteomic and phosphoproteomic data revealed that high temperatures regulated signal transduction, energy metabolism, protein synthesis, cell survival and apoptosis, and cell cytoskeleton remodeling by affecting protein content and phosphorylation levels. Differential phosphorylation modifications of protein kinases A (PKA), extracellular signal-regulated kinase 1 (ERK1), Src tyrosine kinase (SRC), and AKT were identified as potential hub regulatory genes, enhancing glycolysis and TCA cycle to increase energy supply, regulating protein synthesis, inhibiting Caspase-dependent apoptosis triggered by endogenous mitochondrial cytochrome C release, and maintaining cell cytoskeleton stability to mediate C. angulata high heat tolerance. This study comprehensively evaluated the differential dynamic cellular changes in response heat stress between C. gigas and C. angulata at multiple molecular regulatory levels, uncovering numerous key genes and phosphorylation sites mediating their heat resistance differentiation.
Subsequent investigations focused on the phosphorylation functionality and their upstream regulatory pathways of key enzymes involved in glycolysis, pyruvate kinase (PK), the core regulatory factor of NF-κB, inhibitor of NF-κB alpha (IκBα), and the critical executor of cell apoptosis, caspase-3 (Caspase3).
①The phosphorylation levels of IκBα Ser74 in C. gigas and C. angulata exhibited significant differences during heat stress, with upregulation in C. angulata and downregulation in C. gigas. Functional phosphorylation experiments revealed that this site could independently mediate its ubiquitin-proteasome degradation and decrease thermal stability. This mechanism differs from the established cascade mechanisms in model organisms which IKKs kinase phosphorylates IκBα Ser32 and Ser36 to control its protein degradation. This site specifically evolved in oyster major IκBα and is phosphorylated by ERK1/2 kinase, and regulated by the classical MAPK pathway. Differential phosphorylation of oyster IκBα under heat stress promoted stronger nuclear entry of REL1 and REL2 in C. angulata, activating the expression of genes involved in cell survival, fatty acid metabolism, protein translation, and antioxidation, thereby aiding in heat stress resistance. This study firstly reported a novel heat-induced phosphorylation site (Ser74) in the core regulatory factor IκBα of NF-κB, a conserved immune pathway in metazoans. The oyster's high-temperature-RTK-MRAS-BRAF-MAPK-NF-κB signaling cascade exhibited differential heat responses and temperature adaptation patterns between C. gigas and C. angulata, indicating its involvement in shaping the divergent temperature adaptation and heat survival of these two species.
②The phosphorylation levels of Caspase-3/7 Thr260 site in C. gigas and C. angulata exhibited significant differences during heat stress, with upregulation in C. angulata  and downregulation in C. gigas. Based on evolutionary analysis and sequence alignment, a group of novel Caspase-3/7 genes with a long interdomain linker region (IL) in the Bivalvia was discovered. Further in vivo and in vitro functional experiments revealed that the long IL can inhibit the cleavage activation of Bivalvia novel CASP3/7. The Thr260 site located within the IL region is conserved in the class Bivalvia and is phosphorylated by AKT kinase, thereby suppressing the heat-induced activation of CASP3/7. The phosphorylation of the Thr260 site was regulated by the classical PI3K-AKT pathway and exhibited divergent heat response patterns between C. gigas and C. angulata, indicating the involvement of the species-specific PI3K-AKT-CASP3/7 signaling cascade in mediating the divergent heat-induced apoptosis between these two species. This study firstly reported the PI3K-AKT-CASP phosphorylation regulatory pathway in non-model organisms, providing new insights into the complex and unique apoptotic mechanisms in mollusks.
③The phosphorylation levels of PK Ser11 site in C. gigas and C. angulata exhibited significant differences during heat stress, with higher upregulation observed in C. angulata compared to C. gigas. The Ser11 site is conserved in the class Bivalvia and Gastropoda. Phosphorylation at this site enhanced substrate binding, thereby increasing PK enzyme activity and promoting glycolysis to enhance ATP synthesis, aiding in the heat damage repair of oysters. Further functional validation of its upstream regulatory pathway revealed that ERK1/2 kinase phosphorylated PK Ser11 site, which was regulated by the classical MAPK pathway. The mollusk-specific high-temperature-RTK-MRAS-BRAF-MAPK-PK signaling cascade (Bivalvia and Gastropoda) exhibited differential heat responses and temperature adaptation patterns between C. gigas and C. angulata. This suggests that phosphorylation, by regulating the rate of glycolysis, affects the divergent energy metabolism capabilities under heat stress, thereby mediating the divergent heat resistance between these two species.
These findings highlight the existence of complex and unique phosphorylation-mediated regulatory networks of conserved pathways in marine organisms, which mediate the differential temperature adaptation and heat tolerance between two congeneric oyster species, C. gigas (a relatively heat-sensitive species) and C. angulata (a relatively heat-tolerant species) by influencing cell survival, apoptosis, and energy metabolism. The results of this study further expand our understanding of the evolution and function of the crosstalk mechanisms among established classical pathways. Additionally, the identified heat-resistant key effector genes, upstream regulatory factors, and their corresponding phosphorylation sites provide a theoretical basis and important targets for genetic improvement of heat resistance traits in oyster industry.

第1章 绪论    1
1.1 温度适应性性状的研究进展    2
1.1.1 长期适应—脂肪酸    6
1.1.2 短期响应—耐热性    8
1.2 多层级分子调控网络介导差异温度适应    10
1.2.1 遗传水平    10
1.2.2 转录水平    11
1.2.3 蛋白及蛋白翻译后水平    12
1.3 海洋生物脂肪酸和耐热性研究现状    13
1.4 长牡蛎和福建牡蛎的温度适应性分化    14
1.4.1 总体概况    14
1.4.2 脂肪酸    15
1.4.3 耐热性    16
1.5 本研究的目的和意义    16
第2章 温度适应性性状分化    18
2.1 研究背景    18
2.2 材料与方法    18
2.2.1 同质化养殖    18
2.2.2 急性热应激实验    19
2.2.3 生长数据测量    19
2.2.4 营养性状测量    19
2.2.5 抗氧化能力测量    20
2.2.6 非胁迫及热胁迫状态呼吸率测量    23
2.2.7 热胁迫鳃组织形态变化    23
2.2.8 热胁迫鳃组织TUNEL染色    24
2.2.9 热胁迫鳃组织抗氧化能力测量    24
2.2.10 统计分析    26
2.3 实验结果    26
2.3.1 生长数据差异    26
2.3.2 营养性状差异    27
2.3.3 抗氧化能力差异    28
2.3.4 非胁迫及热胁迫状态呼吸率差异    28
2.3.5 热胁迫状态鳃组织形态学变化    29
2.3.6 热胁迫状态鳃组织凋亡率差异    30
2.3.7 热胁迫状态鳃组织抗氧化能力差异    31
2.4 讨论    32
2.4.1 长牡蛎和福建牡蛎之间存在生长-防御权衡    32
2.4.2 相对低温适应的长牡蛎倾向于储存糖原和脂肪酸    32
2.4.3 相对高温适应的福建牡蛎进化出更强的耐热性    33
2.5 本章小结    33
第3章 脂肪酸性状关键基因分化机制    34
3.1 研究背景    34
3.2 材料与方法    34
3.2.1 实验材料    34
3.2.2 总RNA提取    34
3.2.3 cDNA文库构建和测序    35
3.2.4 序列比对和差异表达基因分析    35
3.2.5 代谢物提取和检测    35
3.2.6 差异代谢物分析    36
3.2.7 转录组和代谢组联合分析    37
3.2.8 RNAi干扰实验    37
3.2.9 酿酒酵母过表达实验    37
3.2.10 荧光漂白恢复（FRAP）实验    40
3.2.11 细胞Western Blotting实验    42
3.2.12 双荧光素报告酶实验    42
3.2.13 Scd启动子区顺式调控变异鉴定    43
3.2.14 Scd启动子区顺式调控变异位点功能鉴定    44
3.2.15 DNA Pull-Down实验    45
3.2.16 EMSA实验    47
3.2.17 短期冷胁迫实验    49
3.2.18 转录因子调控关系验证    49
3.2.19 杂交F2群体独立验证    49
3.2.20 统计分析    50
3.3 实验结果    53
3.3.1 长牡蛎和福建牡蛎转录组分析    53
3.3.2 长牡蛎和福建牡蛎代谢组分析    55
3.3.3 长牡蛎和福建牡蛎转录组代谢组联合分析    58
3.3.4 牡蛎Scd基因功能验证    61
3.3.5 长牡蛎和福建牡蛎Scd启动子区顺式调控变异鉴定    64
3.3.6 长牡蛎和福建牡蛎Scd顺式调控变异功能验证    67
3.3.7 长牡蛎和福建牡蛎短期冷胁迫期间Scd的可塑性模式及调控    68
3.3.8 顺式调控变异杂交F2群体独立验证    70
3.4 讨论    72
3.4.1 长牡蛎福建牡蛎在多性状相关基因和代谢物存在分化    72
3.4.2 长牡蛎福建牡蛎在油酸含量和Scd表达上呈现分化可塑性模式    75
3.4.3 顺式调控变异（Cis-Varations）介导Scd基础表达差异    76
3.4.4 反式调控变异（Trans-Varations）介导Scd塑性表达差异    76
3.5 本章小结    78
第4章 基于多组学的耐热性性状分化机制研究    80
4.1 研究背景    80
4.2 材料与方法    80
4.2.1 实验样品    80
4.2.2 总RNA提取    80
4.2.3 cDNA文库构建和测序    81
4.2.4 ATAC-Seq文库构建    81
4.2.5 RNA-Seq和ATAC-Seq数据分析    81
4.2.6 HSF1及其亚型的qRT-PCR实验    82
4.2.7 蛋白提取和胰酶消化    82
4.2.8 IMAC富集    83
4.2.9 LC-MS/MS分析    83
4.2.10 蛋白组和磷酸化组数据分析    84
4.2.11 统计分析    84
4.3 实验结果    84
4.3.1 长牡蛎和福建牡蛎热胁迫差异表达基因    84
4.3.2 长牡蛎和福建牡蛎热胁迫差异染色质可及性    87
4.3.3 ATAC-Seq和RNA-Seq的联合分析    91
4.3.4 蛋白质组和磷酸化组数据概述    97
4.3.5 蛋白质组和磷酸化组生信分析结果    99
4.3.6 鉴定介导差异耐热性的关键DEPs和DPPs    103
4.4 讨论    107
4.4.1 福建牡蛎可能进化出集中且高效的热响应调控网络    107
4.4.2 细胞凋亡和能量代谢是介导二者差异耐热性的关键效应通路    108
4.4.3 差异蛋白和磷酸化调控多细胞过程介导福建牡蛎高耐热性    111
4.5 本章小结    116
第5章 NF-κB/IκB磷酸化介导的差异热存活机制    118
5.1 研究背景    118
5.2 材料与方法    118
5.2.1 实验样品    118
5.2.2 系统发育分析    119
5.2.3 牡蛎IκB基因表达分析    121
5.2.4 分子进化分析和祖先重建    121
5.2.5 Co-IP    121
5.2.6 亚细胞定位    122
5.2.7 双分子荧光互补（BiFC）实验    122
5.2.8 双荧光素报告酶实验    122
5.2.9 细胞核细胞质蛋白分离    123
5.2.10 免疫组化（IHC）实验    123
5.2.11 EMSA实验    123
5.2.12 细胞凋亡检测实验    123
5.2.13 细胞活性（CCK-8）实验    124
5.2.14 RNAi干扰实验    124
5.2.15 RNAi后RNA-Seq    124
5.2.16 DAP-Seq    124
5.2.17 差示扫描荧光实验（DSF）    125
5.2.18 体内泛素化实验    125
5.2.19 酵母双杂交    125
5.2.20 激酶预测    126
5.2.21 体内磷酸化实验    126
5.2.22 体外磷酸化实验    127
5.2.23 qRT-PCR    127
5.2.24 Western blotting    127
5.2.25 统计分析    128
5.3 实验结果    130
5.3.1 牡蛎IκBα及其Ser74的分子进化分析    130
5.3.2 牡蛎主效IκBα基因功能    134
5.3.3 牡蛎IκBα Ser74位点磷酸化功能    137
5.3.4 牡蛎REL热胁迫介导的下游效应基因    141
5.3.5 牡蛎CgERK1/2与CgIκBα S74的激酶-底物互作验证    143
5.3.6 牡蛎MAPK/CgERK1/2T187 Y189与NF-κB通路的调控关系验证    146
5.3.7 牡蛎MAPK/ERK/IκBα/REL调控轴的环境响应性    150
5.4 讨论    152
5.4.1 牡蛎主效IκBα特异性进化的Ser74磷酸化独立介导其泛素降解    152
5.4.2 牡蛎中存在高温-RTK-MRAS-BRAF-MAPK-NF-κB信号级联    153
5.4.3 牡蛎MAPK/NF-κB信号级联高温下差异激活细胞存活等下游基因    154
5.5 本章小结    155第6章 Caspase/Caspase-3/7磷酸化介导的差异热凋亡机制    156
6.1 研究背景    156
6.2 材料与方法    157
6.2.1 实验样品    157
6.2.2 系统发育分析    157
6.2.3 牡蛎Caspase3/7基因表达分析    164
6.2.4 体外CASP酶活检测    164
6.2.5 体内CASP3酶活检测    165
6.2.6 细胞活性（CCK-8）实验    165
6.2.7 细胞凋亡检测实验    165
6.2.8 LDH释放检测实验    165
6.2.9 细胞TUNEL染色    165
6.2.10 激酶预测    166
6.2.11 Co-IP    166
6.2.12 双分子荧光互补（BiFC）实验    166
6.2.13 酵母双杂交    166
6.2.14 亚细胞定位    166
6.2.15 体内磷酸化实验    166
6.2.16 体外磷酸化实验    166
6.2.17 Western blotting    166
6.2.18 统计分析    166
6.3 实验结果    168
6.3.1 双壳纲新型Caspase-3/7系统发育分析    168
6.3.2 双壳纲新型Caspase-3/7长IL功能验证    172
6.3.3 双壳纲新型Caspase-3/7 IL T260磷酸化功能    174
6.3.4 AKT与双壳纲新型Caspase-3/7IL 的激酶-底物互作验证    176
6.3.5 PI3K-AKT调控双壳纲新型Caspase-3/7 IL T260磷酸化验证    178
6.4 讨论    182
6.4.1 双壳纲进化出具有长IL的新型Caspase-3/7    182
6.4.2 双壳纲新型Caspase-3/7的IL区域的Thr260磷酸化抑制裂解激活    182
6.4.3 PI3K-AKT介导双壳纲新型Caspase-3/7 Thr260磷酸化    183
6.5 本章小结    185
第7章 Glycolysis/PK磷酸化介导差异的热能量代谢机制    186
7.1 研究背景    186
7.2 材料与方法    186
7.2.1 实验样品    186
7.2.2 系统发育分析    186
7.2.3 PK酶活检测    188
7.2.4 PK相关代谢物检测    188
7.2.5 葡萄糖摄取速率检测    189
7.2.6 糖酵解速率检测    190
7.2.7 分子动力学模拟    190
7.2.8 表面等离子共振（SPR）检测    191
7.2.9 激酶预测    192
7.2.10 Co-IP    192
7.2.11 双分子荧光互补（BiFC）实验    192
7.2.12 酵母双杂交    192
7.2.13 亚细胞定位    192
7.2.14 体内磷酸化实验    193
7.2.15 体外磷酸化实验    193
7.2.16 Western blotting    193
7.2.17 统计分析    193
7.3 实验结果    194
7.3.1 软体动物PK系统发育分析及序列比对    194
7.3.2 CgPK S11磷酸化功能验证    195
7.3.3 CgPK S11磷酸化介导的PK-底物结合能力评估    197
7.3.4 牡蛎CgERK1/2与CgPK S11的激酶-底物互作验证    203
7.3.5 牡蛎MAPK/CgERK1/2T187 Y189与Glycolysis/PK调控关系验证    205
7.3.6 牡蛎MAPK/ERK/PK调控轴的环境响应性    207
7.4 讨论    209
7.4.1 牡蛎PK Ser11磷酸化通过增强K279活性口袋提升其酶活    209
7.4.2 牡蛎MAPK/ERK1/2磷酸化PK Ser11参与牡蛎温度适应性分化    210
7.5 本章小结    210
全文总结    212
创新点、不足与展望    215
参考文献    217
致  谢    254
作者简历及攻读学位期间发表的学术论文与其他相关学术成果    257