实验海洋生物学重点实验室知识库

  牡蛎是世界大宗养殖的海水贝类，是人类可利用的重要海洋渔业资源之一，为全球性分布种类。长牡蛎（Crassostrea gigas）作为我国华北地区重要的海水养殖品种，肉鲜味美，具有很高的药用价值和营养价值，深受消费者喜爱。但近年来高密度养殖和全球环境变化严重危害了牡蛎养殖业的健康发展。随着养殖规模的不断扩大，长牡蛎因病害导致大面积死亡的事件层出不穷，成为了限制养殖可持续发展的关键因素。

  牡蛎为固着生活在潮间带的滤食性动物。尽管缺乏适应性免疫系统，长牡蛎却通过复杂高效的天然免疫系统适应了存在多样病原的潮间带环境。长牡蛎基因组的解析发现了大量生理防御系统基因家族的显著扩张，可能是适应复杂多变环境的新机制。其中，免疫防御基因C1qDC家族被认为是扩张最为显著的基因家族之一。但目前C1qDC基因家族的分子演化历程及其免疫防御功能的分化轨迹尚不清楚。测序技术的迅速发展为我们解析该问题提供了有力支撑。本研究拟结合生物信息分析、应激转录组测序和原位杂交等技术，明确C1qDC基因家族成员的序列特征和时空表达模式，阐释C1qDC基因家族的分子演化及防御功能分化轨迹。本研究的主要结果如下：

1. 本研究利用生物信息学的方法注释了44个后生动物基因组中的C1qDC基因家族成员，通过基因家族演化分析，确定了C1qDC基因家族在软体动物中的显著扩张事件；根据系统发育树追溯了扩张事件的起源时间，发现了软体动物中C1qDC基因家族呈现谱系特异性扩张，明确了软体动物门中腹足纲和双壳纲的C1qDC基因的扩张事件是相对独立的协同演化；同义突变（Ks）分布曲线表明长牡蛎C1qDC基因家族可能扩张了3次，且大部分基因由近期扩张事件而来；染色体上的定位分析表明串联重复是C1qDC基因扩张的主要方式，且若干复制过程为演化近期发生。这些结果鉴定了C1qDC基因家族的序列特征，描述了其分子演化历程。

2. 下载NCBI数据库中不同发育时期、成体不同组织以及响应不同胁迫因子时的转录组数据，计算C1qDC家族成员的基因表达量，发现长牡蛎中特异扩张的C1qDC基因呈现消化腺特异表达模式。在此基础上，选取长牡蛎三种组织（消化腺、鳃和血细胞），分别进行生物胁迫环境弧菌、病毒类似物和化学胁迫环境重金属的刺激，文库构建和转录组测序。生物信息学分析结果表明C1qDC基因家族成员能够被生物和化学胁迫环境胁迫表达。值得注意的是，扩张的不同成员已经发生了组织特异性应激表达模式分化。根据加权基因共表达网络分析（WGCNA），结果表明不同组织和非生物胁迫条件下存在作为候选中枢基因的C1qDC基因。这些结果表明了长牡蛎扩张的C1qDC基因呈现组织特异的功能分化，阐述了其功能演化轨迹。

3. 为了进一步明确长牡蛎C1qDC基因的组织特异性表达模式，本研究从软体动物门双壳纲的三个C1qDC基因的扩张亚群中选取了30个目标基因，进行长牡蛎整胚或组织原位杂交实验。通过原位探针的信号，可以确定在整胚原位杂交实验中的8个C1qDC基因和组织原位杂交实验中的12个C1qDC基因呈现组织特异表达模式。值得注意的是，这20个C1qDC基因均呈现出消化腺器官（如：肝胰脏、肠道）特异的空间表达模式。此外，针对外套膜特异表达的C1qDC基因，本研究利用透射电镜实验对长牡蛎外套膜细胞进行了超微结构的观察，探究了C1qDC基因与生物矿化的相关性。这些结果明确了长牡蛎C1qDC基因的空间表达模式，为后续功能验证研究奠定了基础。

  本研究阐明了长牡蛎C1qDC基因家族的扩张事件，明确了其表达分化演化和防御功能分化轨迹，为软体动物先天免疫和抗逆研究提供数据基础，也为长牡蛎抗病育种和开发海洋新型活性物质提供一定的理论支撑。

    As one of the important marine fishery resources available to human beings, the oyster is a globally distributed species. Crassostrea gigas, as an important marine breeding variety in North China, has delicious meat, high medicinal value and rich nutrients, and is deeply loved by consumers. However, in recent years, high-density cultivation and global environmental changes have seriously jeopardized the healthy and sustainable development of oyster cultivation. With the continuous expansion of the cultivation scale, the death of bacterial or viral diseases of C. gigas occurred frequently, and gradually became the key limiting factor of its cultivation.

    Oysters are filter feeders that live fixedly in the intertidal zone. Despite the lack of an adaptive immune system, C. gigas has adapted to the intertidal environment with a variety of pathogens through a complex and efficient natural immune system. The analysis of the theme revealed the significant expansion of a large number of gene families of phytate in the biological defense system, which may be a new mechanism to adapt to the complex and changeable environment. Among them, the immune defense C1qDC gene family is considered to be one of the most significant gene families in its expansion. However, the molecular evolution of the C1qDC gene family and the differentiation trajectory of immune defense function remain unclear. The rapid development of sequenced technology provides strong support for us to solve this problem. In this study, the sequence characteristics and spatio-temporal expression patterns of the C1qDC gene family members were determined by combining bioinformatics analysis, RNA-seq, in situ hybridization, molecular evolution, and differentiation trajectory of defense function of the C1qDC gene family were elucidated. The main results of this study are as follows:

1. In this study, the C1qDC gene family members in 44 metazoan genomes were annotated using bioinformatics methods, and significant expansion events of the C1qDC gene family in mollusks were determined through gene family evolution analysis. The phylogenetic tree was used to trace the origin of the expansion event, and it was found that the C1qDC gene family showed lineage-specific expansion in mollusks, and it was confirmed that the expansion event of the C1qDC gene in gastropods and bivalves in mollusks was relatively independent coevolution. The Ks distribution curve showed that the C1qDC gene family expanded three times, and most of the genes were derived from recent expansion events. Chromosome location analysis showed that tandem duplication was the main mode of the C1qDC gene expansion, and several replication processes occurred recently. These results identified the sequence characteristics of the C1qDC gene family and described its molecular evolution.

2. Transcriptome data of different developmental stages, different adult tissues, and responses to different stress factors were downloaded from the NCBI database, and gene expression levels of the C1qDC family members were calculated. It was found that the specific expanded C1qDC gene in C. gigas showed a digestive gland specific expression pattern. On this basis, three tissues (digestive glands, gills, and hemolymph) of C. gigas were selected for stimulation of vibrio, viral analogs, and heavy metals under biological stress, respectively. Library construction and transcriptome sequencing were performed. Bioinformatics analysis showed that C1qDC gene family members could be induced by biological and chemical stress. Notably, different members of the expansion have undergone tissue-specific stress expression pattern differentiation. WGCNA analysis showed that there were candidate hub gene of C1qDC gene under different tissues and stress conditions. These results indicated that the expanded C1qDC gene of C. gigas showed tissue-specific functional differentiation and explained its functional evolution trajectory.

3. To further clarify the tissue-specific expression pattern of the C1qDC gene in C. gigas, 30 target genes were selected from three extended subpopulations of the C1qDC in the bivalve of mollusks phylum for in situ hybridization of whole embryos or tissues of C. gigas. Through the signal of the RNA probes, there were 8 C1qDC genes in the whole embryo in situ hybridization experiment and 12 C1qDC genes in the tissue in situ hybridization experiment identified to show the tissue-specific expression patterns. Notably, 20 C1qDC genes showed a specific spatial expression pattern of digestive gland (e.g., hepatopancreas and intestines). In addition, transmission electron microscopy was used to observe the ultrastructure of the C1qDC gene specifically expressed in mantle of C. gigas in this study, and the correlation between the C1qDC gene and biological mineralization was explored. These results clarified the spatial expression pattern of the C1qDC gene in C. gigas and laid a foundation for subsequent functional verification studies.

    This study elucidates the expansion event of the C1qDC gene family in C. gigas and clarifies the differentiation and evolution of the C1qDC gene family and the differentiation trajectory of defense function, providing data basis for the study of innate immunity and resistance to stress in mollusks, and theoretical support for disease-resistant breeding of C. gigas and development of new marine active substances.

目 录

第1章 绪 论... 1

1.1       免疫系统概述... 2

1.1.1    天然免疫与适应性免疫... 2

1.1.2    无脊椎动物的免疫识别... 3

1.1.3    C1q免疫识别功能的演化... 4

1.2       基因复制概述... 6

1.2.1    红皇后理论... 6

1.2.2    基因家族扩张的生态学意义... 7

1.3       C1qDC基因家族的研究现状... 9

1.3.1    C1qDC基因在生物胁迫中的作用... 10

1.3.2    C1qDC基因在非生物胁迫中的作用... 12

1.4       本研究目的和意义... 13

第2章 C1qDC基因家族序列鉴定与特征分析... 15

2.1       引言... 15

2.2       材料与方法... 15

2.2.1    C1qDC基因家族的鉴定... 15

2.2.2    结构域预测... 16

2.2.3    构建基因系统发育树和物种系统发育树... 16

2.2.4    信号肽预测和染色体定位分析... 17

2.2.5    基因复制事件分析... 17

2.3       结果... 17

2.3.1    C1qDC基因家族在物种中的鉴定... 17

2.3.2    C1qDC基因家族扩张的时间估算... 21

2.3.3    C1qDC基因家族在染色体上的分布... 26

2.4       讨论... 28

2.4.1    C1qDC基因家族的鉴定... 29

2.4.2    C1qDC基因家族扩张的时间估算... 29

2.4.3    C1qDC基因家族在染色体上的分布... 30

第3章 长牡蛎C1qDC基因表达模式分析... 32

3.1       引言... 32

3.2       材料与方法... 32

3.2.1    构建基因系统发育树... 32

3.2.2    转录组数据获取... 32

3.2.3    样品收集... 33

3.2.4    RNA提取... 34

3.2.5    RNA-seq文库构建及测序... 34

3.2.6    测序数据质量控制和表达量计算... 35

3.2.7    差异基因（DEGs）的鉴定和分析... 35

3.2.8    加权共表达网络（WGCNA）的构建与分析... 35

3.3       结果... 36

3.3.1    RNA-seq测序结果... 36

3.3.2    长牡蛎转录组数据的分析... 36

3.3.3    长牡蛎C1qDC基因在组织上的特异表达... 38

3.3.4    长牡蛎C1qDC基因在重金属和病原胁迫下的差异表达    41

3.3.5    加权共表达网络分析... 47

3.4       讨论... 51

3.4.1    长牡蛎C1qDC基因在组织上的特异表达... 51

3.4.2    长牡蛎C1qDC基因在重金属和病原胁迫下的差异表达    52

3.4.3    长牡蛎在重金属和病原胁迫下的基因网络分析... 55

第4章 长牡蛎C1qDC基因空间表达模式鉴定... 56

4.1       引言... 56

4.2       材料与方法... 56

4.2.1    引物设计... 56

4.2.2    整胚原位杂交... 58

4.2.3    组织原位杂交... 64

4.2.4    苏木精-伊红染色（H.E染色）... 66

4.2.5    长牡蛎外套膜透射电镜实验... 66

4.3       结果... 67

4.3.1    C1qDC基因在长牡蛎不同发育时期的消化腺组织定位    67

4.3.2    C1qDC基因在长牡蛎成体消化腺组织中的定位... 70

4.4       讨论... 78

总结与展望... 81

参考文献... 85

附录I 主要试剂... 97

致 谢... 99

作者简历及攻读硕士期间发表的学术论文及研究成果... 101