中国科学院海洋研究所机构知识库

作为栖息在潮间带的重要经济物种，牡蛎经常受到强烈的温度波动胁迫。近年来，随着全球变暖趋势加剧，栖息在潮间带的生物的生存压力与日俱增。牡蛎的夏季大规模死亡现象对产业造成巨大经济影响，其被认为与高温胁迫有关，因此研究牡蛎热适应机制具有重要生态和产业意义。本研究选取具有明显的地理分布和热适应能力差异的巨蛎属两姊妹亚种—长牡蛎（Crassostrea gigas gigas）和福建牡蛎（C. gigas angulata），通过对HSF1在牡蛎热激过程中的作用及其与HSP之间的调控关系的了解来阐释牡蛎热耐受性差异的调控机制。我们首先克隆鉴定热激响应的HSF1不同亚型；而后通过转录组（HSF1干扰后的热应激）和染色质免疫沉淀后测序（ChIP-seq）研究HSF1在热激中作用及其下游调控基因；之后在两姊妹亚种（C. gigas gigas和C. gigas angulata）中比较了HSP基因序列差异以及其与HSF1调控关系；最后通过对一个在两亚种中具有表达差异的HSP70基因的启动子序列研究，来阐释牡蛎中复杂的热适应调节机制。

1.热激条件下响应的HSF1亚型

为了解福建牡蛎和长牡蛎哪种HSF1的可变剪切形式在热激响应中会发挥主要作用，我们在热激后的材料中进行克隆，并在不同热激程度的材料中进行亚型的表达模式分析。结果表明在长牡蛎和福建牡蛎中主要有两种HSF1亚型（HSF1a和HSF1d）响应热应激，且它们在不同的温度应激下的表达丰度不同。而后对两亚种的HSF1a和HSF1d亚型分别进行亚细胞定位实验，发现它们都可以在热激后从均匀分布的状态到聚集到细胞核内点状分布。结合相互作用实验表明HSF1d可以相互结合形成聚体。本部分的实验结果表明在两亚种中响应于热应激表达的HSF1亚型可能都参与了热激调控。

2. 通过转录组和基因干扰技术对长牡蛎热激响应中HSF1的调控研究

为了研究在热激状况下的响应基因以及受HSF1调控的基因的情况，本研究处理得到以下四组材料：对照组、35 ℃-2 h热激组、HSF1干扰组和干扰后热激组。将这四种材料进行转录组测序，转录组分析结果得到了150个热激响应差异基因，其中包括七个HSP基因。加权基因共表达网络分析（WGCNA）得到的17个HSP基因中有六个也通过差异基因富集得到与热激相关。此外，我们发现在热激中，被HSF1上调和下调的基因分别是48和47个。在上调的基因中，我们得到了一个可能受HSF1响应热激调控的HSP70基因（CGI_10002387）。通过结合位点预测和荧光素酶双报告基因实验初步证实了HSF1对富集到的HSP70的调控作用。基于差异表达的基因和WGCNA分析，在热激条件下富集到了低氧信号通路。此外，在热激中，HSP70和HSP20是最早响应的。

3. 通过染色质免疫共沉淀测序技术研究HSF1下游调控基因

通过染色质水平寻找HSF1在热激中结合的基因，从染色质水平分析HSF1的调控情况。测序得到916个与HSF1结合位点相对应的基因，分析发现有四个HSP70基因，两个HSP40基因和一个小HSP基因表现出与HSF1的结合能力。在GO分析中，HSF1的靶基因与信号转导，能量产生以及对生物刺激的反应有关。并且验证了一个在启动子区域具有结合位点的HSP70（CGI_10003417）在热激下受HSF1调控。

4. 长牡蛎和福建牡蛎中差异表达的HSP序列比较及其与HSF1关系研究

为探究长牡蛎和福建牡蛎热激下基因响应差异原因，对两亚种中表达差异显著的HSP基因进行序列分析及其与HSF1调控关系的研究。序列分析结果显示两亚种中对应的HSP基因编码的蛋白质序列高度相似，且具有相同的功能域，表明这些HSP基因的功能在两亚种中是保守的。然而，启动子区域序列在两亚种中存在较大差异。对差异较大的两个基因分析发现在两亚种中的表达模式相似，可能具有相同的调控模式。随后通过荧光素酶双报告实验研究HSF1和HSP的调控关系，发现HSF1d亚型在长牡蛎中的激活作用比在福建牡蛎中强，HSF1a则表现为在福建牡蛎中更强的激活作用。启动子区差异最大的基因被激活的程度最高，且在两亚种中被调控的程度差异最大。说明这个基因（CGI_10002594）的启动子区序列的差异可能是造成热激状态下两亚种表达程度差异的重要原因。

5. 对HSP70（CGI_10002594）基因的启动子区域序列研究

通过分析启动子区域序列情况，试图解析序列差异在调控关系中的地位。分析发现序列变异类型包括片段的插入或缺失（indel）以及单碱基突变，福建牡蛎具有更多indel的个体。indel多是富含AT的重复序列，在两亚种的差异序列相似度较高。推测位点突变（A/G）可以使序列与HSF1的结合潜力增加，且发现indel和A/G突变存在关联。连锁不平衡分析结果富含AT的序列indel和A/G突变表现为很高的连锁不平衡特性，且A/G突变基因型在两亚种群体比较中呈现出显著差异。随后对不同单倍型进行调控关系研究表明，单倍型II被激活的程度更高。综上，该位点变异和富含AT的indel序列可能影响了基因的表达。

总而言之，我们的研究表明长牡蛎可能存在HSF1-HSP调控关系。且结果显示，在牡蛎的热激调控中似乎存在更加复杂的机制。本实验的探索有利于对牡蛎耐热性机制的了解，并为耐热性的标记辅助育种提供候选基因和理论支持，从而为牡蛎的夏季大规模死亡研究提供参考。

Oyster, a commercially important species inhabiting in the intertidal zone, often exposed to temperature fluctuations. More stress pressure was put on the organisms inhabiting the intertidal zone with the global warming process going on in recent years. Summer mortality, which lead to great loss of economy and was thought be related to thermal stress. Thus, it is meaningful to study the mechanism of thermal stress in oysters. The materials, the Pacific oyster (Crassostrea gigas gigas) and Fujian oyster (C. gigas angulata), which with significantly differences in geographical distribution and ability to adapt to thermal stress, were used to illustrate the thermal stress mechanisms in oyster by identifying the role of HSF1 and the regulatory relationship between HSPs under heat shock stress. Firstly, we cloned the HSF1 isoforms which play an important role in heat shock response in C. gigas gigas and C. gigas angulata. Then, we performed transcriptomic (thermal stress following HSF1 interference), chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the Pacific oyster. We compared the sequences variation and regulatory relationship between HSF1 and HSP in two congener oyster species (C. gigas gigas and C. gigas angulata). Those studies were used to explain the complex thermal adaptation regulation mechanism in oysters.

1. Isoforms of HSF1 under heat shock conditions in oyster

To determine which isoforms of HSF1 play an important role response to thermal stress in Fujian oyster and the Pacific oyster, we cloned the sequences and quantified gene expression level of isoforms in the heat-shocked materials. We found that there are two mainly isoforms of HSF1 (HSF1a and HSF1d) responding to heat shock in two subspecies and they expression differently under various thermal stress. Then, subcellular localization of two subtypes was performed and the results show that that distribution of both subtypes transformed from the dispersed state to points gathering in the nucleus when exposed to thermal stress. Interaction experiments show that they can be formed aggregates. Those results show that HSF1 isoforms which response to heat shock could contribute regulation in heat shock in Fujian oyster and the Pacific oyster.

2. RNAi based transcriptome for HSF1 regulatory relationship study in the Pacific oyster under heat shock

To determine the response genes and the genes regulated by HSF1 under heat shock, the oysters was separated to four groups, the control group, the heat shock group, the HSF1 RNAi group and the heat shock following RNAi group. Four groups were used to do RNA sequencing; the results of transcriptome analysis identified 150 genes responsive to heat shock including seven HSP genes. Six of 17 HSP genes enriched in response to heat shock according to weighted gene co-expression network analysis (WGCNA). In addition, we found that 48 and 47 genes were up- regulated and down-regulated by HSF1 in response to heat shock, respectively. Among the up-regulated genes, we found a HSP70 (CGI_10002387), which may be regulated by HSF1 in response to heat shock. We identified and confirmed one HSP70 potentially regulated by HSF1 in response to heat shock by binding site predictions and a dual luciferase assay. Hypoxia signaling pathways were enriched under heat shock conditions by differentially expressed genes and WGCNA analysis. In addition, our results revealed that the expression of HSP70 and HSP20 was initially triggered after 2-h of heat shock.

3. Identify the HSF1 regulated genes in the Pacific oyster under thermal stress by ChIP-seq

To sequence the fragment of binding sites in chromatin level which related to genes regulation are easier to find the gene regulated by HSF1 under heat shock condition. In the study, we analyzed the genes from the ChIP-seq results. The genes were obtained by peaks (916 peaks corresponding to HSF1 binding sites) annotated. The results showed that there were four HSP70 genes, two HSP40 genes and one small HSP gene that showed binding to HSF1. In Gene Ontology analysis, HSF1 target genes were related to signal transduction, energy production, and response to biotic stimulus. One HSP70 (CGI_10003417) with a binding site in the promoter region was validated to be regulated by HSF1 under heat shock.

4. Sequences comparison of differentially expressed HSPs and the regulatory relationship with HSF1 in the Pacific oyster and Fujian oyster

To investigate the reasons that differential gene expression level in response to heat shock in the Pacific oyster and Fujian oyster, sequence of the HSPs (with significant differences in expression) and their regulatory relationship with HSF1 in two subspecies were studied. The results of HSP sequence analysis showed that the proteins encoded by the corresponding HSP genes were highly similar and the functional domain were similar in two subspecies. It indicates that the functions of these HSP genes are conserved in both subspecies. The differences were found in the promoter sequences comparation. The degree of difference of HSPs was calculated by comparing the sequences in the promoter region. Analysis of the expression patterns of the two genes with large differences found that the expression patterns in the two subspecies are similar and may have the same regulatory relationship. Then, the regulatory relationship between HSF1 and HSPs was studied by Dual luciferase assay. It showed that genes with the largest differences in promoter regions were activated the most. Moreover, the active of isoforms changes most between the two subspecies. The results indicate that the difference in the promoter region sequence of the gene (CGI_10002594) may contribute to the difference in expression levels of the two subspecies under heat shock.

5. The promoter region Sequence of HSP70 (CGI_10002594) Gene

The sequence of the promoter region was used to analyze the role of sequence differences in regulatory relationships. The sequence analysis showed that there existed the sequence variation including insertion, deletion (indel) and SNP. And the Fujian oyster with more sequences with indel than the Pacific oyster. Furthermore, the indels were AT-rich repeats and were similar in both subspecies. Site mutation (A/G) can increase the possibility of sequence bond by HSF1, and it is found that there is a correlation between the fragments and A/G mutation. The results of linkage disequilibrium analysis showed that a high linkage disequilibrium characteristic of the AT-rich sequences and A/G mutations existed in the two subspecies. Moreover, the A/G mutant genotypes showed significantly differences in the comparison of the two subspecies. The Dual-luciferase assay showed haplotype II are more capable of being activated. In summary, the A/G site mutation and indel which contains AT sequence may affect gene expression.

Overall, the results revealed a possible HSF1–HSP regulatory relationship in the oyster. The results show that a more complex mechanism of heat shock regulation may exist in oysters than model species. The study may provide valuable information on the mechanisms of thermal tolerance and may provide insights into marker assistant breeding for thermal tolerance to possibly avoid summer mass mortality in this commercially important species.

第1章  绪论... 1

1.1  为什么要研究牡蛎热应激？... 1

1.1.1  牡蛎资源保护面临挑战... 2

1.1.2  牡蛎夏季大规模死亡现象... 2

1.2  生理响应机制研究... 4

1.2.1  重要的热激响应分子... 5

1.2.2  热激响应主要调控关系研究进展... 7

1.2.3  调控通路研究方法... 9

1.3  遗传多态性对生物热耐受的影响... 10

1.4  本研究的目的及意义... 11

第2章  长牡蛎和福建牡蛎中HSF1功能研究... 13

2.1  研究背景... 13

2.2  材料和方法... 13

2.2.1  活体材料和热激处理... 13

2.2.2  引物信息... 14

2.2.3  基因全长扩增及序列分析... 15

2.2.4  不同可变剪切形式在热激下的表达情况检测... 15

2.2.5  构建用于细胞实验的表达质粒... 17

2.2.6  细胞培养、传代及质粒转染... 18

2.2.7  热激前后细胞内HSF1分布检测... 19

2.2.8  HSF1蛋白互作研究... 20

2.3  结果... 21

2.3.1  热激状态下福建牡蛎HSF1两种亚型的分析... 21

2.3.2  温度应激下两亚种中不同亚型具有不同的表达模式... 27

2.3.3  两亚种中不同可变剪切形式特征分析... 28

2.3.4  不同可变剪切形式在热激下亚细胞定位变化... 29

2.3.5  免疫共沉淀实验揭示长牡蛎HSF1亚型可形成聚体... 30

2.4  讨论... 31

第3章  基于RNAi的转录组揭示热激下长牡蛎中可能受HSF1调控的基因... 33

3.1  研究背景... 33

3.2  材料和方法... 33

3.2.1  活体材料和预实验处理... 33

3.2.2  预实验效果检测-HSF1表达水平... 35

3.2.3  正式实验材料处理... 35

3.2.4  通过免疫印方法对蛋白定量... 35

3.2.5  转录组测序... 36

3.2.6  转录组数据分析... 37

3.2.7  DEGs可靠性验证... 39

3.2.8  统计分析... 41

3.3  结果... 41

3.3.1  预实验确定样品处理方法... 41

3.3.2  通过RNA干扰实验筛选用于测序样品... 43

3.3.3  转录组数据分析... 44

3.3.4  热激响应转录本... 47

3.3.5  可能受HSF1调节的热激响应的差异基因（DEGs）... 49

3.3.6  基因共表达网络分析... 51

3.3.7  关键响应基因验证... 60

3.4  讨论... 62

第4章  通过ChIP-seq鉴定长牡蛎热激响应中HSF1的下游基因... 67

4.1  研究背景... 67

4.2  材料和方法... 67

4.2.1  活体材料和处理方法... 67

4.2.2  热激转录因子1（HSF1）的蛋白表达水平检测... 67

4.2.3  ChIP-seq数据分析... 68

4.2.4  主要基因的表达模式分析... 68

4.2.5  基因调控作用研究... 69

4.3  结果... 69

4.3.1  HSF1检测... 69

4.3.2  ChIP-seq数据分析... 71

4.3.3  对ChIP鉴定的HSF调控相关基因进行注释... 71

4.3.4  HSF特异性结合位点和调控基因分析... 72

4.3.5  靶基因的表达模式和调控关系... 74

4.4  讨论... 76

第5章  基于候选基因的两亚种热适应差异的研究... 79

5.1  研究背景... 79

5.2  材料和方法... 79

5.2.1  活体材料... 79

5.2.2  牡蛎呼吸率测定... 79

5.2.3  基因序列扩增及分析... 80

5.2.4  荧光实时定量揭示特定基因的表达模式... 82

5.2.5  荧光素酶双报告实验检测热激蛋白基因被HSF1调控情况... 82

5.3  结果... 83

5.3.1  应对热应激时两亚种呼吸率水平存在差异... 83

5.3.2  基因全长及启动子区域差异分析... 84

5.3.3  表达模式研究... 86

5.3.4  荧光素酶双报告实验检测热激蛋白受调控情况... 87

5.4  讨论... 89

第6章  热激蛋白基因（CGI_10002594）启动子序列研究... 91

6.1  研究背景... 91

6.2  材料和方法... 91

6.2.1  活体材料... 91

6.2.2  启动子区域特定位点基因频率研究... 91

6.2.3  荧光素酶双报告检测片段受调控情况... 92

6.3  结果... 93

6.3.1  特定基因区域群体水平基因序列分析... 93

6.3.2  不同序列受调控情况比较... 98

6.4  讨论... 99

第7章  结论和展望... 101

参考文献... 103

附录... 119

致谢... 145

作者简历及攻读学位期间发表的学术论文与研究成果... 147

作者简历：... 147

已发表学术论文：... 147