| 其他摘要 | 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. |
| 目录 | 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 |
修改评论