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硬壳蛤应对高温低氧胁迫的响应特征研究
胡志
Subtype博士
Thesis Advisor张涛
2023-05-15
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Keyword硬壳蛤 高温 低氧 生理响应 分子调控
Abstract

硬壳蛤Mercenaria mercenaria于1997年从美国引种至我国,经过20余年的示范推广,已成为我国北至辽宁、南至广西沿海池塘生态混养的重要经济贝类。硬壳蛤具有很强的抗高温和抗低氧能力:成贝耐受温度达35℃;在0.2mg/L极端低氧条件下7天存活率接近100%。自然环境中高温低氧往往不是两个独立的事件,当水温升高时,氧溶解度降低,高温低氧胁迫往往同时发生。硬壳蛤成贝运动能力较弱,只能通过分子调控网络调整其自身生理和生化状态来抵御环境胁迫。因此探究硬壳蛤应对高温低氧胁迫的响应特征,可以为硬壳蛤健康养殖和遗传育种提供数据支撑和理论依据,同时也为双壳贝类生态适应性进化研究提供基础数据。
本研究利用Realtime PCR、比色法、RNA-seq、UPLC-MS/MS以及生物信息学等技术探究了硬壳蛤应对高温低氧胁迫的响应特征。从能量代谢和抗氧化应激方面查明了硬壳蛤应对高温低氧胁迫的生理响应特征。从转录水平、代谢水平和脂质代谢水平揭示了硬壳蛤应对高温低氧胁迫的分子调控机制,筛选出了关键的通路和差异表达基因及代谢物。利用生物信息学技术,在硬壳蛤基因组中鉴定了HSP70和MAPKK基因家族,并进一步查明了其在硬壳蛤应对高温低氧胁迫的表达特征。研究发现,硬壳蛤应对低氧胁迫时,能够调节基因表达和代谢物含量相对稳定,维持机体稳态。硬壳蛤应对协同胁迫的响应特征与单独高温胁迫类似。在高温、低氧和协同胁迫下,硬壳蛤都会通过增强无氧代谢缓解能量供应不足。而在高温和协同胁迫下,硬壳蛤还会增强糖酵解供应能量,积累氨基酸含量增加对胁迫的耐受力,并通过尿素循环缓解氨/氮毒性。在高温、低氧和协同胁迫下,硬壳蛤都会激活抗氧化系统,增加T-AOC能力缓解氧化应激,并维持较为稳定的MDA水平。此外,在高温和协同胁迫下有机渗透剂和维生素等小分子代谢物,戊糖磷酸途径也在缓解氧化应激中发挥着重要作用。抗凋亡可能是硬壳蛤应对短期高温低氧胁迫的核心转录调控机制。在高温和协同胁迫下,硬壳蛤还会上调分子伴侣相关基因、积累热保护剂缓解蛋白质结构损伤,增强内质网蛋白加工和泛素介导蛋白水解维持稳态。可能通过调节甘油磷脂类、甘油脂类和鞘脂类代谢缓解细胞膜损伤。具体研究结果如下:
1、生理响应特征
从亚细胞、分子和生化水平研究了硬壳蛤在高温低氧胁迫下的能量响应特征。在高温、低氧和协同胁迫下,硬壳蛤鳃线粒体受到损伤,出现去极化。在低氧胁迫下,线粒体分裂标记基因fis1和线粒体自噬标记基因pgam5表达量显著增加,而在高温及协同胁迫下,线粒体融合标记基因mfn2和opa1表达量显著降低。硬壳蛤分别通过促进线粒体分裂和自噬,以及抑制线粒体融合,来维持线粒体功能。在高温、低氧及协同胁迫下,乳酸脱氢酶酶活力以及无氧代谢标志物琥珀酸和乳酸含量显著增加,表明硬壳蛤通过增强无氧代谢缓解能量供应不足。除此之外,糖酵解关键限速酶(磷酸果糖激酶、丙酮酸激酶)酶活力以及糖酵解关键中间代谢物(磷酸二羟丙酮、磷酸烯醇丙酮酸、丙酮酸)含量在高温和/或协同胁迫下显著增加,表明硬壳蛤还会通过增强糖酵解供应能量。多种氨基酸以及尿素循环中间代谢物含量在高温及协同胁迫后显著增加,表明氨基酸含量的积累增加了硬壳蛤对高温及协同胁迫的耐受力,并通过尿素循环缓解氨/氮毒性。通过Realtime PCR、LC-MS和比色法测定了抗氧化酶和非酶抗氧化剂变化特征。为了缓解氧化应激,在高温、低氧及协同胁迫下,硬壳蛤激活抗氧化系统,增加T-AOC能力,并维持较为稳定的MDA水平,但采取了不同的抗氧化策略。在低氧胁迫下,GSH起着重要的抗氧化保护作用。在高温胁迫下,SOD酶活力显著升高。在协同胁迫下,硬壳蛤通过增强SOD、CAT和GST酶活力缓解氧化应激。在高温和协同胁迫下,戊糖磷酸途径在缓解氧化应激中也发挥着重要作用。
2、转录调控机制
通过转录组测序,获得了对照组、高温、低氧及协同胁迫下的mRNA表达谱。PCA结果显示,低氧组和对照组表达模式较为接近,而高温组和协同组表达模式较为接近,常温组(对照组和低氧组)与高温组(高温组和协同组)明显分离。凋亡相关通路在低氧胁迫后显著富集,抗凋亡基因(IAP)表达量显著上调,凋亡执行者(Caspase 8和Caspase 3)表达量显著降低,硬壳蛤可能通过抑制细胞凋亡,防止细胞不必要的大量死亡,以应对低氧胁迫。蛋白质折叠是高温胁迫下最显著富集的GO条目,绝大多数分子伴侣基因(如HSP90,HSP60,HSP40,TCP1等)表达量显著上调,硬壳蛤通过上调分子伴侣相关基因缓解蛋白质结构损伤。内质网蛋白加工和泛素介导蛋白水解途径在高温及协同胁迫下显著富集,GSEA结果表明内质网蛋白加工和泛素介导蛋白水解途径在高温及协同胁迫下得到增强,硬壳蛤可能通过增强内质网蛋白加工和泛素介导蛋白水解途径缓解高温及协同胁迫带来的损伤。凋亡是共有差异表达基因显著富集的通路,多条IAP基因在胁迫后表达量上调,抗凋亡可能是硬壳蛤应对短期高温低氧胁迫的核心转录调控机制。
3、代谢调控机制
通过广泛靶向代谢组学方法,在硬壳蛤鳃组织中共鉴定出了810种代谢物,并筛选差异代谢物。在高温、低氧及协同胁迫下,糖酵解和TCA循环等主要供能途径的中间代谢物含量发生显著变化,无氧代谢产物(琥珀酸、延胡索酸、乳酸)和肉碱含量显著积累提示硬壳蛤通过增强无氧代谢和脂肪酸β氧化来供应能量。甘油磷脂类代谢物在高温及协同胁迫后含量显著增加。在高温及协同胁迫下,有机渗透剂和维生素等差异代谢物可能有助于缓解ROS应激。此外,单糖、氧化三甲胺等热保护剂的显著积累有助于维持蛋白质稳态。
4、脂质代谢调控机制
通过广泛靶向脂质组学方法,对高温、低氧、协同胁迫及对照组硬壳蛤鳃组织中的脂质代谢物进行定性定量检测,共鉴定出了913种脂质代谢物。甘油脂类、甘油磷脂类和鞘脂类是最主要的差异脂质代谢物。绝大多数差异脂质代谢物,在胁迫后含量显著积累。硬壳蛤可能通过甘油磷脂代谢、甘油脂类代谢、鞘脂类代谢等通路改变鳃组织的脂质组成。
5、HSP70基因家族鉴定及其在高温低氧胁迫下的表达特征
基于硬壳蛤全基因组序列,使用序列相似性(Blastp)和保守结构域相似性(HMMER)并结合手动矫正方法,在硬壳蛤基因组中鉴定了133条硬壳蛤MmHSP70基因。MmHSP70基因不均等的分布在19条染色体和4条scaffolds上,其中41条MmHSP70基因位于第7号染色体上。系统发育分析显示,MmHSP70蛋白主要分为两大枝,HSP70 12亚家族发生了大规模扩张。共线性分析显示硬壳蛤Mercenaria mercenaria第7号染色体与青蛤Cyclina sinensis第14号染色体存在一个高密度的HSP70共线性区块。获得和丢失分析显示,硬壳蛤获得了62条HSP70基因。串联重复是MmHSP70基因大规模扩张的主要驱动力之一,对串联重复MmHSP70基因对进行选择压力(Ka/Ks)分析,发现其经历了不同程度的纯化选择。而且相同串联重复基因对的MmHSP70具有相似的基因结构和motif。大多数MmHSP70串联重复基因对在常温组(对照组和低氧组)中表达量较高,MmHSP70 B2串联重复基因对在协同胁迫下表达量显著增加。
6、MAPKK基因家族鉴定及其在高温低氧胁迫下的表达特征
基于硬壳蛤全基因组序列,使用Blastp和HMMER相结合的方法,在硬壳蛤基因组中鉴定了5条MmMAPKK基因。MmMAPKK基因分布在三条染色体上。虽然不同双壳贝类的基因组大小差异很大(如硬壳蛤M. mercenaria基因组1.79 Gb,长牡蛎Crassostrea gigas基因组586.8 Mb),但MAPKK基因数量相对稳定。双壳贝类MAPKK蛋白系统发育分析显示,MAPKK分为5个clades,而且相同clade具有相似的motif模式。共线性分析显示硬壳蛤与青蛤有4对MAPKK同源基因对。选择压力分析结果表明硬壳蛤和青蛤MAPKK基因Ka/Ks值均显著小于0.1,表明受到了强烈的纯化选择。在高温胁迫下,MAPKK7、MAPKK4、ERK3和p38表达量显著上调。在协同胁迫下,MAPKK7、MAPKK6、MAPKK4和p38表达量显著上调。结果表明MAPKK4/MAPKK6-p38级联反应可能在硬壳蛤应对高温及协同胁迫中发挥重要作用。

Other Abstract

The hard clam Mercenaria mercenaria was introduced to our country from America in 1997. After more than 20 years of promotion, it has become an important economic shellfish of ecological poly-culture in coastal ponds from Liaoning province to Guangxi province in our country. The hard clam has strong resistance to heat and hypoxia: the adult clam could tolerate temperature up to 35℃, the survival rate was close to 100% at 7 days under 0.2mg/L extreme hypoxia stress. In the natural environment, heat and hypoxia are often not two independent events. When the water temperature increase, the solubility of oxygen decrease, heat commonly accompany with hypoxia. Due to its weak moveability, the adult hard clam can only adjust their physiological and biochemical states through molecular regulatory networks to cope with environmental stress. Therefore, understanding the response characteristics of hard clam to heat and hypoxia stress can provide data support and theoretical basis for the healthy culture and genetic breeding of the hard clam, and also provide basic data for the study of ecological adaptive evolution of bivalve. 
In this study, Realtime PCR, colorimetry, RNA-seq, UPLC-MS/MS and bioinformatics techniques were used to investigate the response characteristics of hard clam to heat and hypoxia stress. The physiological responses of the hard clam to heat and hypoxia stress were investigated from the aspects of energy metabolism and antioxidant stress. The molecular regulatory mechanism of the hard clam response to heat and hypoxia stress were revealed from the transcriptomic, metabolomic and lipidomic level, and the key pathways and differential expression genes and metabolites were also screen out. Through bioinformatics, HSP70 and MAPKK gene families in the hard clam genome were identified, and their expression characteristics in the hard clam response to heat and hypoxia stress were also explored. The results showed that the hard clam can regulate gene expression and metabolite content relatively stable to maintain homeostasis under hypoxia stress. The response characteristics of the hard clam to combined stress were similar to those of single heat stress. The hard clam alleviated energy deficit by enhancing anaerobic metabolism under heat, hypoxia and combined stress. The hard clam also enhanced glycolysis to supply energy, accumulated free amino acid content to increase the stress tolerance, and alleviated ammonia/nitrogen toxicity through urea cycle under heat and combined stress. The hard clam activated antioxidant system, increased T-AOC capacity to alleviate oxidative stress, and maintained a relatively stable MDA level under heat, hypoxia and combined stresses. In addition, small molecule metabolites (organic osmolytes and vitamins) and pentose phosphate pathway also played a vital role in alleviate oxidative stress under heat and combined stress. Anti-apoptosis may be the core transcriptional regulation mechanism of hard clam coping with short-term heat and hypoxia stress. Under heat and combined stress, the hard clam might also alleviate protein structure damage by up-regulating molecular chaperone related genes and accumulating heat protections, maintain homeostasis by enhancing Protein processing in endoplasmic reticulum and Ubiquitin mediated proteolysis. The hard clam might relieved cell membrane damage through Glycerophospholipid metabolism, Glycerolipid metabolism and Sphingolipid metabolism. The specific findings are as follows:
1. Physiological response characteristics
The energy metabolism response characteristics of the hard clam to heat and hypoxia stress were studied at subcellular, molecular and biochemical levels. Under heat, hypoxia and combined stress, the mitochondria of hard clam gills were impaired and depolarization. Under hypoxia stress, the expressions of mitochondrial fission marker gene (fis1) and mitophagy marker gene (pgam5) were significantly increased. While under heat and combined stress, mitochondrial fusion marker genes mfn2 and opa1 were significantly decreased. The hard clam maintained mitochondrial function by promoting mitochondrial fission and mitophagy, and inhibiting mitochondrial fusion, respectively. Lactate dehydrogenase enzyme activity and anaerobic metabolism biomarkers: succinic acid and lactate content, were significantly increased under heat, hypoxia and combined stress, which indicating that the hard clam alleviated energy deficit by enhancing anaerobic metabolism. In addition, the enzyme activities of key rate-limiting enzymes (phosphofructokinase, pyruvate kinase) and the content of key intermediate metabolite of glycolysis (dihydroxyacetone phosphate, phosphoenolpyruvic acid, pyruvic acid) were significantly increased under heat and/or combined stress, which indicated that the hard clam also enhanced glycolysis to supply energy. The contents of multiple amino acids and urea cycle intermediate metabolites increased significantly under heat and combined stress, which indicated that the hard clam accumulated amino acid content to increase the stress tolerance, and alleviated ammonia/nitrogen toxicity through urea cycle under heat and combined stress. The characteristics of antioxidant enzymes and non-enzymatic antioxidants were studied by Realtime PCR, LC-MS, and colorimetry. To alleviate oxidative stress, the hard clam activated antioxidant system, increased T-AOC capacity and maintained a relatively stable MDA level under heat, hypoxia and combined stresses, but adopted different strategies. Under hypoxia stress, GSH played an important role in antioxidant protection. The SOD activity was significantly increased under heat stress. The hard clam could alleviate oxidative stress by enhancing SOD, CAT and GST enzyme activities under combined stress. Pentose phosphate pathway also played a vital role in alleviate oxidative stress under heat and combined stress. 
2. Regulatory mechanism of transcriptomics
Through transcriptome sequencing, the mRNA expression profiles under control group, heat, hypoxia and combined stress were obtained. PCA results showed that the expression patterns of the hypoxia group and the control group were similar, while the expression patterns of the heat group and the combined group were similar. And the normal temperature group (control group and hypoxia group) and the high temperature group (heat group and combined group) were obviously separated. The apoptosis-related pathways were significantly enriched after hypoxia stress, the expression of anti-apoptotic genes (IAP) were significantly up-regulated, and the expression of apoptotic agents (Caspase 8 and Caspase 3) were significantly decreased. The hard clam might prevent unnecessary cell death by inhibiting cell apoptosis to cope with hypoxia stress. Protein folding was the most significantly enriched GO item under heat stress. Most molecular chaperone genes (e.g. HSP90, HSP60, HSP40, TCP1, etc.) were significantly up-regulated. The hard clam might alleviate protein structure damage by up-regulating molecular chaperone related genes. Protein processing in endoplasmic reticulum and Ubiquitin mediated proteolysis were significantly enriched under heat and combined stress. GSEA analysis showed that these two pathways were enhanced under heat and combined stress. The hard clam might alleviate the damage caused by heat and combined stress through enhancing Protein processing in endoplasmic reticulum and Ubiquitin mediated proteolysis. Apoptosis was significantly enriched in common differential expressed genes, multiple IAP genes were up-regulated after stress. The anti-apoptosis may be the core transcriptional regulatory mechanism of hard clam to cope with short-term heat and hypoxia stress. 
3. Regulatory mechanism of metabolomics
A total of 810 metabolites were identified in the gill tissue of hard clam by widely targeted metabolomics approach. And differential expression metabolites were screened. The content of intermediate metabolites in major energy supply pathways such as glycolysis and TCA cycle changed significantly under heat, hypoxia and combined stress. The significant accumulation of anaerobic metabolites (succinic acid, fumaric acid, lactic acid) and carnitine suggested that the hard clam might provide energy by enhancing anaerobic metabolism and fatty acid β-oxidation under heat, hypoxia and combined stress. The contents of glycerophospholipid metabolites increased significantly under heat and combined stress. The significantly differential expression metabolites of organic osmolytes and vitamins might relieve ROS stress under heat and combined stress. Moreover, accumulation of thermos-protective osmolytes (monosaccharide, Trimethylamine N-oxide) were helpful to maintain protein homeostasis.
4. Regulatory mechanism of lipidomics
Through widely targeted lipidomics approach, lipid metabolites in the gill tissues of the hard clam under heat, hypoxia, combined stress and the control group were qualitatively and quantitatively detected, and a total of 913 lipid metabolites were identified. Glycerolipids, glycerophospholipids and sphingolipid were the main differential expression lipid metabolites. The vast majority of differential expression lipid metabolites were significantly accumulated after stress. The hard clam might change the lipid composition of gill tissue through Glycerophospholipid metabolism, Glycerolipid metabolism and Sphingolipid metabolism.
5. Identification of HSP70 gene family and these expression pattern under heat and hypoxia stress
Based on the hard clam genome sequence, 133 MmHSP70 genes were identified using sequence similarity analysis (Blastp), conserved domain similarity analysis (HMMER) and combined with manual filtration. The MmHSP70 genes were unequally distributed on 19 chromosomes and 4 scaffolds, of which 41 MmHSP70 genes were located on chromosome 7. Phylogenetic analysis showed that MmHSP70 protein mainly consisted of two clusters, and HSPa 12 subfamily underwent massive expanded. Synteny analysis showed that there was a high-density HSP70 collinear block between Mercenaria mercenaria chromosome 7 and Cyclina sinensis chromosome 14. Gain and loss analysis revealed that the hard clam obtained 62 HSP70 genes. Tandem duplication was one of the main driving forces for the large-scale expansion of MmHSP70 genes. The selection pressure (Ka/Ks) analysis of tandem duplication MmHSP70 pairs showed that they had undergone different degrees of purifying selection. Moreover, MmHSP70 genes in the same tandem duplication gene pair had similar gene structure and motif. Most of the MmHSP70 tandem duplication gene pairs were highly expressed in the normal temperature group (control group and hypoxia group), and the MmHSP70 B2 tandem duplication gene pairs were significantly up-regulated under combined stress.
6. Identification of MAPKK gene family and these expression pattern under heat and hypoxia stress
Based on the hard clam genome sequence, five MmMAPKK genes were identified using Blastp combined with HMMER. The MmMAPKK genes were distributed on three chromosomes. Although the genome size was highly variable among different bivalve mollusks (e.g., Mercenaria mercenaria 1.79 Gb and Crassosois gigas 586.8 Mb), the number of MAPKK genes was relatively stable. Phylogenetic analysis of MAPKK protein in bivalves showed that MAPKK could be divided into five clades, and the same clade had similar motif patterns. Synteny analysis showed that there were four MAPKK homologous gene pairs between M. mercenaria and C. sinensis. The selection pressure analysis results showed that the Ka/Ks value of MAPKK genes of M. mercenaria and C. sinensis were significantly less than 0.1, suggesting that they were subjected to strongly purifying selection. Under heat stress, the expressions of MAPKK7, MAPKK4, ERK3 and p38 were significantly up-regulated. Under combined stress, the expressions of MAPKK7, MAPKK6, MAPKK4 and p38 were significantly up-regulated. These results indicated that the MAPKK4/MAPKK6-p38 cascade may play an important role in the response to heat and combined stress in the hard clam.

MOST Discipline Catalogue理学 ; 理学::海洋科学
Language中文
Table of Contents

第1章 绪论 1

1.1 前言 1

1.2 双壳贝类应对高温胁迫的响应特征 1

1.2.1 生理响应特征 1

1.2.2 分子调控机制 4

1.3 双壳贝类应对低氧胁迫的响应特征 6

1.3.1 行为适应策略 6

1.3.2 生理响应特征 6

1.3.3 分子调控机制 8

1.4 双壳贝类应对高温低氧协同胁迫的响应特征 10

1.5 本研究的目的、意义与研究思路 11

1.5.1 目的及意义 11

1.5.2 科学问题 11

1.5.3 研究内容及技术路线 11

1.5.4 预期成果 12

第2章 高温低氧胁迫下硬壳蛤生理响应特征 13

2.1 研究背景 13

2.2 材料与方法 13

2.2.1 实验材料和胁迫实验 13

2.2.2 样品收集 14

2.2.3 线粒体膜电位检测 14

2.2.4 RNA提取及荧光定量PCR 14

2.2.5 生化指标检测 15

2.2.6 代谢物含量测定 15

2.2.7 统计分析 15

2.3 实验结果 15

2.3.1 存活率 15

2.3.2 线粒体响应特征 16

2.3.3 能量代谢响应特征 17

2.3.4 抗氧化应激响应特征 20

2.4 讨论 23

2.5 本章小结 26

第3章 高温低氧胁迫下硬壳蛤转录调控机制 27

3.1 研究背景 27

3.2 材料与方法 28

3.2.1 实验材料和样品收集 28

3.2.2 RNA提取 28

3.2.3 文库构建和转录组测序 28

3.2.4 比对到参考基因组和基因表达分析 28

3.2.5 基因差异表达分析 28

3.2.6 GO、KEGG和GSEA富集分析 28

3.3 实验结果 29

3.3.1 转录组测序概况 29

3.3.2 基因表达量 29

3.3.3 差异表达基因分析 30

3.3.4 GO和KEGG功能富集分析 31

3.3.5 基因集富集分析 38

3.4 讨论 40

3.5 本章小结 43

第4章 高温低氧胁迫下硬壳蛤代谢调控机制 45

4.1 研究背景 45

4.2 材料与方法 45

4.2.1 实验材料和样品收集 45

4.2.2 代谢组测序 45

4.2.3 代谢组生物信息学分析 46

4.3 实验结果 46

4.3.1 代谢组表达谱 46

4.3.2 差异代谢物筛选 49

4.3.3 KEGG富集分析 51

4.3.4 高温低氧胁迫下代谢响应的系统分析 52

4.4 讨论 54

4.4.1 糖酵解、无氧代谢和TCA循环 54

4.4.2 脂质代谢 55

4.4.3 有机渗透剂 56

4.4.4 维生素 56

4.4.5 其他代谢反应 57

4.5 本章小结 57

第5章 高温低氧胁迫下硬壳蛤脂质代谢调控机制 59

5.1 研究背景 59

5.2 材料与方法 59

5.2.1 实验材料和样品收集 59

5.2.2 脂质代谢物提取 59

5.2.3 脂质组测序 60

5.2.4 脂质组生物信息学分析 60

5.2.5 转录组数据再挖掘 60

5.3 实验结果 60

5.3.1 脂质组表达谱 60

5.3.2 差异脂质代谢物筛选 63

5.3.3 KEGG富集分析 64

5.3.4 脂质代谢基因分析 66

5.4 讨论 69

5.5 本章小结 72

第6章 HSP70基因家族鉴定及其在高温低氧胁迫下的表达特征 73

6.1 研究背景 73

6.2 材料与方法 73

6.2.1 MmHSP70基因家族鉴定和序列分析 73

6.2.2 基因组定位,基因结构和保守motif特征分析 74

6.2.3 序列比对和系统发育分析 74

6.2.4 共线性分析 74

6.2.5 获得与丢失分析 75

6.2.6 高温低氧胁迫下MmHSP70基因表达模式分析 75

6.3 实验结果 75

6.3.1 HSP70基因家族鉴定及序列分析 75

6.3.2 HSP70基因的染色体分布和重复类型 75

6.3.3 HSP70蛋白系统发育分析 78

6.3.4 硬壳蛤与青蛤HSP70基因共线性分析 79

6.3.5 获得与丢失分析 80

6.3.6 串联重复MmHSP70基因在高温低氧胁迫下的表达模式 81

6.4 讨论 82

6.5 本章小结 84

第7章 MAPKK基因家族鉴定及其在高温低氧胁迫下的表达特征 87

7.1 研究背景 87

7.2 材料与方法 88

7.2.1 MmMAPKK基因家族鉴定和序列分析 88

7.2.2 MmMAPKK基因组定位和基因结构分析 89

7.2.3 序列比对和系统进化分析 89

7.2.4 保守motif分析 89

7.2.5 共线性分析 89

7.2.6 选择压力分析 89

7.2.7 不同组织MmMAPKK基因表达模式分析 89

7.2.8 高温低氧胁迫下MmMAPKK及下游MAPK基因表达模式分析 90

7.3 实验结果 90

7.3.1 MAPKK基因家族鉴定及序列分析 90

7.3.2 MmMAPKK基因的染色体分布和基因结构 91

7.3.3 系统发育和保守motif分析 93

7.3.4 共线性分析 94

7.3.5 选择压力分析 95

7.3.6 MAPKK基因在不同组织的表达模式 96

7.3.7 MAPKK及下游MAPK基因在高温低氧胁迫下的表达模式 96

7.4 讨论 97

7.5 本章小结 99

第8章 研究总结与展望 101

8.1 研究总结 101

8.2 主要创新点 102

8.3 存在问题 102

8.4 研究展望 103

参考文献 105

附录一 荧光定量PCR所用的引物 129

附录二 硬壳蛤HSP70基因在染色体上的分布 131

致  谢 133

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 137

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/181193
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
胡志. 硬壳蛤应对高温低氧胁迫的响应特征研究[D]. 中国科学院海洋研究所. 中国科学院大学,2023.
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202018006812067胡志-20(34628KB)学位论文 暂不开放CC BY-NC-SA
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