海洋生态与环境科学重点实验室知识库

硬壳蛤（Mercenaria mercenaria），隶属于软体动物门（Mollusca）、双壳纲（Bivalvia）、帘蛤目（Veneroida）、帘蛤科（Veneridae）、硬壳蛤属（Mercenaria）。硬壳蛤原产自北美大西洋沿岸，从潮间带至水深15米的海底均有分布。自1997年引种至我国以来，经过20余年的推广，硬壳蛤目前已成为我国沿海地区重要的池塘养殖贝类。受持续性降雨、干旱等自然因素和围堤、泄洪等人为因素的影响，陆基养殖池塘的海水会在一定时间内处于高盐或低盐水平。硬壳蛤在长期自然选择中逐渐进化出广盐性，能够调整一系列生理活动和启动复杂精密的分子调控机制来抵抗或适应盐胁迫。探究硬壳蛤应对盐胁迫的抗逆性和适应性生理响应特征和广盐适应性进化机制有利于揭示硬壳蛤对盐度的生态适应和进化适应机制，并为硬壳蛤健康养殖和耐盐良种选育提供数据支撑和理论依据。

本研究探究了硬壳蛤应对盐胁迫的抗逆性和适应性生理响应特征和广盐适应性进化机制。利用广泛靶向代谢组学和生理生化检测技术，查明了硬壳蛤应对急性短期和慢性长期盐胁迫的的抗逆性和适应性生理响应特征；利用RNA-seq和流式细胞检测技术，查明了硬壳蛤应对急性短期和慢性长期盐胁迫的转录调控机制。利用BLASTP和HMMER等生物信息学技术，从硬壳蛤基因组中鉴定出所有的水通道蛋白（AQP）基因家族成员，并查明其结构特征、理化性质、与其他双壳贝类AQP的进化关系和在盐胁迫下的基因表达特征；通过比较基因组学手段，鉴定出硬壳蛤基因组中显著扩张的基因家族和在进化过程中经历正选择的基因，初步查明了硬壳蛤广盐适应性进化机制。具体研究结果如下：

1、硬壳蛤应对急性短期高盐和低盐胁迫的抗逆性生理响应特征

通过广泛靶向代谢组学技术，从急性短期高盐和低盐胁迫下的硬壳蛤鳃组织中共鉴定出979个小分子代谢物。通过K-means分析，鉴定出了硬壳蛤响应急性短期高盐胁迫的关键代谢物（如尿囊素、肉桂酸、葫芦巴碱和L-去甲肾上腺素）。在急性短期高盐胁迫下，己糖激酶和丙酮酸激酶活力显著上升，并且丙酮酸含量显著上升，表明硬壳蛤体内糖酵解过程得到增强。第5天（d）时，丙氨酸和乳酸显著积累于鳃组织中，标志着厌氧糖酵解过程得到增强。与此同时，氨基酸分解代谢增强，导致大量氨积累于鳃组织中。硬壳蛤通过上调谷氨酰胺合成酶和谷氨酸脱氢酶活力的方式减轻过量氨对细胞造成的毒性。许多抗氧化代谢物（如尿囊素、肉桂酸和葫芦巴碱）含量显著上升以缓解氧化损伤。此外，大量溶血磷脂含量显著上升，暗示受到严重氧化损伤的细胞膜脂质被磷脂酶水解，可能有利于维持细胞膜整体的稳定性和渗透性。在急性短期低盐胁迫下，硬壳蛤鳃组织中Na⁺/K⁺-ATP酶（NKA）活力显著上升，Na⁺和Cl^-浓度显著下降。急性低盐胁迫5 d时，环磷酸腺苷（cAMP）含量显著上升。与此同时，腺苷酸环化酶、蛋白激酶A、钠通道蛋白和钙通道蛋白的基因表达水平均大幅下降。该结果表明，cAMP-PKA通路在急性短期低盐胁迫5 d时被抑制，有利于阻止环境中的无机离子进入鳃细胞。NKA酶活力的上调和cAMP-PKA信号通路的抑制很可能是硬壳蛤在急性短期低盐胁迫下调控离子跨膜转运的重要机制。此外，许多抗氧化代谢产物（如丝氨酸和含酪氨酸的二肽）含量均在急性短期低盐胁迫下显著上升，有利于抵抗氧化应激。甘油磷脂代谢过程也得到显著增强，可能有利于稳定细胞膜脂质结构。急性低盐胁迫1 d时，硬壳蛤鳃组织中丙氨酸和乳酸含量显著上升，标志着无氧代谢过程得到增强。此外，大量酰基肉碱显著积累于鳃组织中，表明脂肪酸β氧化过程得到增强，从而有利于为渗透压调控提供能量。总的来说，硬壳蛤在急性短期高盐和低盐胁迫下的抗逆性生理响应特征展现出较高水平的相似性。

2、硬壳蛤应对慢性长期高盐和低盐胁迫的适应性生理响应特征

使用广泛靶向代谢组学技术，从慢性长期高盐和低盐胁迫下的硬壳蛤鳃组织中共鉴定出1225个小分子代谢物。差异代谢物的KEGG功能富集分析结果显示，硬壳蛤在慢性长期高盐和低盐胁迫过程中均通过上调ABC转运蛋白通路以提高氨基酸和小肽的跨膜转运效率，并通过抑制产热作用以减少能量的热损耗。在慢性长期低盐胁迫下，硬壳蛤细胞膜受到严重的氧化损伤。一些细胞保护剂的含量显著上升，可能在增强蛋白质稳定性和缓解氧化损伤上发挥重要作用。甘油磷脂代谢和细胞骨架相关通路在慢性长期低盐胁迫过程中被显著抑制，表明细胞膜和细胞骨架的稳定性受到一定程度的损伤。硬壳蛤在慢性长期高盐和低盐胁迫过程中倾向于使用氨基酸衍生物、小肽和葫芦巴碱等有机渗透剂来调控渗透压。

3、硬壳蛤应对急性短期和慢性长期盐胁迫的转录调控机制

使用RNA-seq技术，探究了硬壳蛤应对急性短期和慢性长期盐胁迫的转录调控机制。在急性短期和慢性长期低盐胁迫下，硬壳蛤通过抑制氨基酸合成和增强氨酰-tRNA合成以降低胞内FAAs含量。在慢性长期高盐胁迫下，大量自噬相关基因（ATGs）的表达量显著上升。借助流式细胞检测技术，发现硬壳蛤鳃细胞自噬活力和自噬率均在慢性高盐胁迫下显著上升。当细胞自噬被NH₄Cl抑制时，鳃组织中总FAAs含量显著下降。该结果表明硬壳蛤在慢性长期高盐胁迫下能够通过激活细胞自噬以增加胞内FAAs含量，进而恢复渗透平衡。此外，细胞自噬的激活还有利于清除胞内受损严重的蛋白和维持内环境稳态。当盐胁迫对硬壳蛤造成的细胞损伤较轻或可修复时，IAPs的表达量显著上升且CTSL的表达量显著下降，以避免细胞凋亡被过早激活。在慢性长期盐胁迫下，多巴胺和去甲肾上腺素的合成显著增强，花生四烯酸和谷胱甘肽代谢通路被显著抑制，表明神经内分泌响应是硬壳蛤应对慢性长期盐胁迫的重要的适应性生理响应。

4、硬壳蛤水通道蛋白基因家族成员鉴定及其在盐胁迫下的基因表达特征

结合BLASTP和HMMER方法，从硬壳蛤基因组中共鉴定出9个AQP基因家族成员。每个MmAQPs均具有6或12个跨膜α-螺旋、1–2个MIP结构域和2个NPA motif。所有的MmAQPs被归类为3个亚家族：类AQP1、类AQP3和类AQP8。在硬壳蛤基因组中并未鉴定出类AQP11亚家族成员。基因组共线性分析结果表明，AQP1、AQP3、AQP4、AQP5和AQP8很可能在帘蛤目物种中是高度保守的。在进化过程中，串联重复事件导致硬壳蛤类AQP8亚家族发生大幅扩张，3个MmAQP8紧密排列于9号染色体上。硬壳蛤类AQP3亚家族在进化过程中可能经历了基因丢失或假基因化。基因表达分析结果表明，各MmAQPs在盐胁迫下的基因表达特征表现出较大差异。例如，MmAQP5_2特异性响应高盐胁迫，MmAQP8_2特异性响应低盐胁迫，MmAQP4则特异性响应慢性长期盐胁迫。串联重复事件很可能推动了MmAQP5和MmAQP8在响应盐胁迫上发生基因功能分化。

5、硬壳蛤广盐适应性进化机制

选择7种广盐性双壳贝类和5种狭盐性双壳贝类进行比较基因组学分析，鉴定出了包括硬壳蛤在内的广盐性双壳贝类中显著扩张的基因家族和正选择基因（PSGs）。广盐性双壳贝类的SLC23基因家族在进化过程中经历了显著扩张，有利于增强其跨膜转运抗坏血酸和清除胞内活性氧物质（ROS）的能力。此外，硬壳蛤体内许多抗氧化酶基因（如GST和TXNRD）经历了适应性进化，且其基因表达水平在慢性长期高盐胁迫下显著上升。硬壳蛤的PSGs显著富集于多条碳水化合物代谢和脂质代谢相关通路，可能有利于增强在盐胁迫下的能量供给效率。PSGs显著富集于跨膜运输和无机阴离子运输等GO条目上。SLC22和离子通道基因经历了正选择，有利于提高硬壳蛤在盐胁迫下的离子跨膜转运效率。硬壳蛤的PSGs还显著富集于多条氨基酸代谢通路，有利于其在盐胁迫下高效地调节胞内FAAs浓度。鞘脂代谢和鞘糖脂生物合成通路均被PSGs显著富集，表明硬壳蛤细胞膜脂质代谢相关通路或生理过程经历了适应性进化。PLA2的适应性进化有利于硬壳蛤在盐胁迫下迅速溶解受损严重的细胞膜，并促进新膜的合成。上述研究结果表明，硬壳蛤体内能量代谢、渗透压调控、ROS清除和细胞膜脂质重塑等生理过程在长期自然选择中均经历了适应性进化。以上显著扩张的基因家族和PSGs构成了硬壳蛤广盐性特性的重要分子基础。

Hard clams (Mercenaria mercenaria), belonging to the phyla Mollusca, class bivalvia, order Veneroida, family Veneridae, and genus Mercenaria. Hard clams are native to the Atlantic coast of North America, distributing in the intertidal zone to the bottom of the sea at depths of 15 meters. After more than 20 years of development since its introduction to China in 1997, hard clams have become an important pond aquaculture bivalve species in coastal areas of China. Under the influence of natural factors such as continuous rainfall and drought and human factors such as embankment and flood discharge, the seawater of land-based aquaculture ponds would be in a state of high or low salinity for a certain period of time. Hard clams have evolved eurysalinity over a long period of natural selection. Hard clams can regulate a series of physiological activities and activate sophisticated molecular regulatory mechanisms to tolerate or adapt to salinity stress. Investigation of the characteristics of resistant and adaptive physiological response to salinity stress and the adaptive evolutionary mechanisms of eurysalinity in hard calms is conducive to revealing the ecological and evolutionary adaptation mechanisms to salinity, and providing data support and theoretical basis for healthy culture and salinity-tolerant breeding of hard clams.

This study investigated the resistant and adaptive physiological response characteristics under salinity stress and the adaptive evolutionary mechanisms of eurysalinity in hard clams. The widely targeted metabolomics and physiological and biochemical assays were used to identify the resistant and adaptive physiological responses of hard clams to acute short-term and chronic long-term salinity stress. RNA-seq and flow cytometry assay were used to determine the transcriptional regulation mechanisms of hard clams to acute short-term and chronic long-term salinity stress. Bioinformatics tools such as BLASTP and HMMER were used to identify all aquaporin (AQP) gene family members in the genome of hard clams. Their structural characteristics, physicochemical properties, evolutionary relationship with other bivalve AQP, and gene expression characteristics under salinity stress were also investigated in hard clams. Through comparative genomics analysis, the significantly expanded gene families and genes that underwent positive selection during evolution were identified in the genome of hard clams. We have preliminarily identified the the adaptive evolutionary mechanisms of eurysalinity in hard clams. The detailed results are as follows:

1. The resistant physiological response characteristics under acute short-term hyper-salinity and hypo-salinity stress in hard clams

A total of 979 small molecule metabolites were identified from the gills of hard clams under acute short-term hyper-salinity and hypo-salinity stress by using widely targeted metabolomics techniques. The critical metabolites (such as allantoin, cinnamic acid, trigonelline and L-norepinephrine) in response to acute short-term hyper-salinity stress were identified using K-means analysis. Under acute short-term hyper-salinity stress, the activities of hexokinase and pyruvate kinase, and the content of pyruvate, were significantly up-regulated, indicating that the glycolysis was enhanced in hard clams. On 5 day (d), alanine and lactic acid were significantly accumulated in gills, indicating that anaerobic glycolysis was enhanced in hard clams. Meanwhile, the amino acid catabolism was enhanced, resulting in the accumulation of large amounts of ammonia in the gills. Hard clams can reduce the cytotoxicity caused by excess ammonia by up-regulating the activity of glutamine synthetase and glutamate dehydrogenase. The relative content of many antioxidant metabolites (such as allantoin, cinnamic acid and trigonelline) were significantly up-regulated to mitigate oxidative damage. The content of many lysophospholipids were significantly up-regulated, implying that severely oxidized cell membrane lipids might be hydrolyzed by phospholipase. This physiological regulation might contribute to maintain the overall stability and permeability of cell membrane. Under acute short-term hypo-salinity stress, Na⁺/K⁺-ATPase (NKA) activity increased significantly, and Na⁺ and Cl^- concentrations decreased significantly in the gills. On 5 d under acute hypo-salinity stress, the relative content of cyclic adenosine phosphate (cAMP) increased significantly. Meanwhile, the gene expression levels of adenylate cyclase, protein kinase A, sodium channel protein, and calcium channel protein were significantly up-regulated. This result suggests that the cAMP-PKA pathway was inhibited on 5 d under acute short-term hypo-salinity stress to prevent the entry of environmental inorganic ions into gill cells. The up-regulation of NKA enzyme activity and the inhibition of cAMP-PKA signaling pathway might be the important mechanisms of ion trans-membrane transport in hard clams under acute short-term hypo-salinity stress. Additionally, many antioxidant metabolites (such as serine and tyrosine-containing dipeptides) were significantly up-regulated under acute short-term hypo-salinity stress to resist oxidative stress. Glycerophospholipid metabolism was also significantly enhanced to stabilize the lipid structure in cell membrane. On 1 d under acute hypo-salinity stress, the contents of alanine and lactic acid in the gills increased significantly, indicating that the anaerobic metabolism was enhanced in hard clams. Additionally, a large amount of acylcarnitines were significantly accumulated in the gills, indicating that β-oxidation of fatty acid was enhanced to provide energy for osmoregulation. In general, a high level of similarity was observed in the resistant physiological response characteristics under acute short-term hyper-salinity and hypo-salinity stress in hard clams.

2. The adaptive physiological response characteristics under chronic long-term hyper-salinity and hypo-salinity stress in hard clams

A total of 1,225 small molecule metabolites were identified from the gills of hard clams under chronic long-term hyper-salinity and hypo-salinity stress using widely targeted metabolomics techniques. KEGG functional enrichment analysis of the differential metabolites showed that the ABC transporter pathway was significantly enhanced to enhance the trans-membrane transport efficiency of amino acids and small peptides. Moreover, thermogenesis was significantly inhibited to reduce the heat loss of energy. Under chronic long-term hypo-salinity stress, the cell membrane of hard clams was seriously damaged by oxidative stress. The significant increase of the content of some cell protectant may play an important role in enhancing protein stability and alleviating oxidative damage. Glycerophospholipid metabolism and cytoskeleton-related pathways were significantly inhibited during chronic long-term salinity stress, suggesting that the stability of cell membrane and cytoskeleton might be damaged. Hard clams tended to use amino acid derivatives, small peptides, trigonelline and other organic osmolytes for osmoregulation under chronic long-term salinity stress.

3. The transcriptional regulation mechanisms of hard clams under acute short-term and chronic long-term salinity stress

RNA-seq was used to explore the transcriptional regulation mechanisms of hard clams in response to acute short-term and chronic long-term salinity stress. Under acute short-term and chronic long-term hypo-salinity stress, amino acid synthesis was significantly inhibited and aminoacyl-trNA synthesis was enhanced to decrease intracellular FAAs content. The expression level of a large number of autophagy related genes (ATGs) was significantly up-regulated under chronic long-term hyper-salinity stress. Flow cytometric analysis showed that the autophagy activity and autophagy rate of gill cells increased significantly under chronic long-term hyper-salinity stress. The total FAAs content in the gills of hard clams decreased significantly after the inhibition of autophagy by NH₄Cl. The above results showed that hard clams could activate autophagy to increase intracellular FAAs content and restore osmotic equilibrium under chronic long-term hyper-salinity stress. The activation of autophagy is also conducive to the clearance of severely damaged proteins and the maintenance of homeostasis of the internal environment. When the cell damage caused by salinity stress was mild or repairable, the expression level of IAPs increased significantly and the expression level of CTSL decreased significantly to avoid premature activation of apoptosis. The synthesis of dopamine and norepinephrine was significantly enhanced, and the metabolic pathways of arachidonic acid and glutathione were significantly inhibited under chronic long-term salinity stress. This suggests that neuroendocrine response is the important adaptive physiological response to chronic long-term salinity stress in hard clams.

4. Genome-wide identification of AQP gene family members in hard clams and their gene expression characteristics under salinity stress

Combined with BLASTP and HMMER methods, a total of 9 AQP gene family members were identified from the genome of hard clams. Each MmAQPs has 6 or 12 trans-membrane alpha helices, 1–2 MIP domains, and 2 NPA motifs. All MmAQPs were classified into three subfamilies: AQP1-like, AQP3-like, and AQP8-like. No member of the AQP11-like subfamily has been identified in the genome of hard clams. Genome synteny analysis showed that AQP1, AQP4, AQP5 and AQP8 might be highly conserved among species belonging to order Venerida. In the course of evolution, tandem duplication events led to a large expansion of the AQP8 subfamily in hard clams, with three MmAQP8 closely arranged on chromosome 9. The AQP3 subfamily in hard clams might experience gene loss or pseudogenization during evolution. Gene expression analyses showed that each MmAQPs exhibited specific gene expression characteristics under salinity stress. For example, MmAQP5_2 specifically responded to hyper-salinity stress. MmAQP8_2 specifically responded to hypo-salinity stress, and MmAQP4 specifically responded to chronic salinity stress. The tandem duplication events might promote the gene functional divergence of MmAQP5 and MmAQP8 in response to salinity stress.

5. The adaptive evolutionary mechanisms of eurysalinity in hard clams

Seven euryhaline bivalves and five stenohaline bivalves were used for comparative genomic analysis. The significantly expanded gene families and positive selection genes (PSGs) were identified in euryhaline bivalves, including hard clams. The SLC23 gene family in euryhaline bivalves has undergone significant expansion during evolution, contributing to improve the ability to transport ascorbic acid across cell membrane and remove intracellular reactive oxygen species (ROS). In addition, the antioxidant genes (such as GST and TXNRD) in hard clams underwent adaptive evolution, and their gene expression level increased significantly under chronic long-term hyper-salinity stress. PSGs in hard clams were significantly enriched in multiple pathways related to carbohydrate metabolism and lipid metabolism, which may be conducive to enhancing the energy supply efficiency under salinity stress. PSGs in hard clams were also significantly enriched in GO terms such as transmembrane transport and inorganic anion transport. The SLC22 and ion channel genes have undergone positive selection in hard clams, which is beneficial to improve the efficiency of ion transmembrane transport under salinity stress. Additionally, PSGs in hard clams was significantly enriched in multiple amino acid metabolism pathways, which is conducive to the efficient regulation of intracellular FAAs concentration under salinity stress. The sphingolipid metabolism and sphingolipid biosynthesis pathways were significantly enriched by PSGs in hard clams, indicating that membrane lipid metabolism-related pathways or physiological processes have undergone adaptive evolution. The adaptive evolution of PLA2 facilitated the rapid dissolution of severely damaged cell membranes and promoted the synthesis of new membranes under salinity stress. The above results indicate that energy metabolism, osmoregulation, ROS scavenging, and cell membrane lipid remodeling in hard clams have undergone adaptive evolution during long-term natural selection. The significantly expanded gene family and PSGs consist of the important molecular basis for the eurysalinity of hard clams.

第1章 绪论 1

1.1 海水盐度变化对双壳贝类的影响 1

1.2 双壳贝类应对盐胁迫的生理响应特征 1

1.2.1 行为响应 1

1.2.2 基础代谢响应 2

1.2.3 能量代谢响应 2

1.2.4 抗氧化响应 3

1.2.5 细胞膜和细胞骨架调整 4

1.2.6 免疫应答和神经内分泌响应 4

1.3 双壳贝类的渗透压调控机制 5

1.3.1 无机离子 5

1.3.2 游离氨基酸 6

1.3.3 水 7

1.4 双壳贝类对盐度的适应性进化 7

1.4.1 双壳贝类的盐度适应范围 7

1.4.2 自然选择 8

1.4.3 适应性进化 8

1.5 硬壳蛤简介 9

1.5.1 硬壳蛤生物学 9

1.5.2 我国硬壳蛤养殖产业现状 10

1.5.3 硬壳蛤生态适应性研究进展 10

1.6 本研究的目的、意义与研究思路 10

1.6.1 目的及意义 10

1.6.2 科学问题 11

1.6.3 研究内容及技术路线 11

1.6.4 预期成果 12

第2章 硬壳蛤应对急性短期盐胁迫的抗逆性生理响应特征 13

2.1 研究背景 13

2.2 材料与方法 14

2.2.1 实验材料和样品收集 14

2.2.2 酶活力和离子浓度检测 15

2.2.3 代谢物提取、鉴定和定量分析 15

2.2.4 差异代谢物鉴定 16

2.2.5 代谢物KEGG注释和功能富集分析 16

2.2.6 K-means分析 16

2.2.7 RNA提取和实时荧光定量PCR 16

2.2.8 统计分析 17

2.3 实验结果 18

2.3.1 急性短期高盐胁迫下酶活力变化规律 18

2.3.2 急性短期低盐胁迫下离子浓度和酶活力变化规律 19

2.3.3 急性短期高盐胁迫代谢组表达谱 21

2.3.4 急性短期低盐胁迫代谢组表达谱 31

2.4 讨论 40

2.4.1 硬壳蛤应对急性短期高盐胁迫的抗逆性生理响应特征 40

2.4.2 硬壳蛤应对急性短期低盐胁迫的抗逆性生理响应特征 43

2.5 本章小结 45

第3章 硬壳蛤应对慢性长期盐胁迫的适应性生理响应特征 47

3.1 研究背景 47

3.2 材料与方法 47

3.2.1 实验材料和样品收集 47

3.2.2 代谢物提取 48

3.2.3 代谢组生物信息学分析 48

3.2.4 统计分析 48

3.3 实验结果 48

3.3.1 代谢物鉴定和样品聚类 48

3.3.2 差异代谢物鉴定 49

3.3.3 差异代谢物的KEGG功能富集分析 53

3.3.4 慢性长期盐胁迫下参与渗透压调控的关键小分子代谢物 55

3.4 讨论 56

3.4.1 硬壳蛤应对慢性长期高盐和低盐胁迫的相似性适应性生理响应特征 56

3.4.2 硬壳蛤应对慢性长期高盐和低盐胁迫的差异性适应性生理响应特征 57

3.5 本章小结 58

第4章 硬壳蛤应对急性短期和慢性长期盐胁迫的转录调控机制 59

4.1 研究背景 59

4.2 材料与方法 60

4.2.1 实验材料和样品收集 60

4.2.2 RNA提取 60

4.2.3 cDNA文库构建和转录组学测序 60

4.2.4 数据质控和基因组映射 60

4.2.5 基因表达水平定量、样品聚类和差异表达分析 61

4.2.6 KEGG功能富集分析 61

4.2.7 加权基因共表达网络分析 61

4.2.8 转录组数据准确性验证 61

4.2.9 自噬活力检测 62

4.2.10 游离氨基酸含量测定 62

4.2.11 统计分析 62

4.3 实验结果 63

4.3.1 转录组测序概况 63

4.3.2 样品聚类和差异表达基因鉴定 63

4.3.3 差异表达基因的KEGG功能富集分析 65

4.3.4 加权基因共表达网络构建和关键模块鉴定 69

4.3.5 实时荧光定量PCR 71

4.3.6 细胞自噬的游离氨基酸调控作用 73

4.4 讨论 76

4.4.1 游离氨基酸浓度调节 76

4.4.2 细胞自噬 77

4.4.3 抗凋亡响应 77

4.4.4 神经内分泌响应 78

4.5 本章小结 79

第5章 硬壳蛤水通道蛋白基因家族成员鉴定 81

5.1 研究背景 81

5.2 材料与方法 82

5.2.1 双壳贝类AQP基因家族成员鉴定 82

5.2.2 双壳贝类AQPs序列比对和系统发育分析 83

5.2.3 MmAQPs的理化性质和亚细胞定位 83

5.2.4 MmAQPs的染色体分布和和基因组内共线性分析 83

5.2.5 MmAQPs的基因结构 83

5.2.6 MmAQPs的蛋白结构 84

5.2.7 MmAQPs与近缘物种AQPs的共线性关系 84

5.2.8 MmAQPs的组织表达谱 84

5.2.9 急性短期和慢性长期盐胁迫下MmAQPs的基因表达特征 84

5.2.10 统计分析 85

5.3 实验结果 85

5.3.1 双壳贝类AQP基因家族成员鉴定和系统发育分析 85

5.3.2 MmAQPs的理化性质 87

5.3.3 MmAQPs的染色体分布和基因组内共线性 87

5.3.4 MmAQPs的基因和蛋白质结构 89

5.3.5 硬壳蛤与近缘物种基因组间共线性的AQP基因对 91

5.3.6 MmAQPs的组织表达谱 92

5.3.7 急性短期和慢性长期盐胁迫下MmAQPs的基因表达特征 93

5.4 讨论 94

5.5 本章小结 96

第6章 硬壳蛤广盐适应性进化机制 97

6.1 研究背景 97

6.2 材料与方法 98

6.2.1 基因组数据收集 98

6.2.2 系统发育树构建和直系同源基因簇鉴定 99

6.2.3 基因家族扩张收缩分析 99

6.2.4 硬壳蛤显中显著扩张的基因家族成员的组织表达谱 99

6.2.5 正选择分析 99

6.2.6 正选择基因的GO和KEGG功能富集分析 100

6.2.7 盐胁迫处理 100

6.2.8 RNA提取和实时荧光定量PCR 100

6.2.9 统计分析 100

6.3 实验结果 101

6.3.1 基因家族聚类和系统发育树构建 101

6.3.2 广盐性双壳贝类中显著扩张和收缩的基因家族 101

6.3.3 硬壳蛤溶质载体家族23的理化性质、结构特征和组织表达谱 103

6.3.4 硬壳蛤正选择基因鉴定和功能富集分析 105

6.3.5 硬壳蛤正选择基因在盐胁迫下的表达特征 107

6.3.6 硬壳蛤广盐适应性进化机制 108

6.4 讨论 109

6.4.1 能量代谢的适应性进化 109

6.4.2 渗透压调控的适应性进化 110

6.4.3 活性氧物质清除的适应性进化 111

6.4.4 细胞膜脂质重塑的适应性进化 111

6.5 本章小结 112

第7章 研究总结与展望 113

7.1 研究总结 113

7.2 主要创新点 115

7.3 存在问题 115

7.4 研究展望 115

参考文献 117

致  谢 139

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 141