实验海洋生物学重点实验室知识库

红螯螯虾（Cherax quadricanatus）俗称澳洲淡水龙虾，是一种重要的新兴经济水产动物，由于其本身的生物学特性可作为甲壳动物的模式生物进行研究。胚胎发育是个体发育的起点，胚胎发育的好坏是影响螯虾体质的重要因素之一。而病原是影响螯虾养殖的另一个重要因素，先天免疫系统是甲壳动物清除和杀死病原的天然防御系统。对螯虾胚胎发育和先天免疫开展研究对于丰富甲壳动物发育模式和抗病研究具有重要的意义，同时也为螯虾养殖的疾病防御提供了重要的指导作用。本研究以红螯螯虾为研究对象，利用转录组和microRNA（miRNA）组学技术对红螯螯虾不同胚胎发育时期进行了研究，同时基于转录组数据对参与先天免疫的C型凝集素和Kazal型丝氨酸蛋白酶抑制因子进行了基因克隆和功能研究。主要研究结果如下：

1、红螯螯虾胚胎发育转录组比较分析

采用Illumina测序技术对红螯螯虾三个胚胎发育时期的转录组谱进行了研究，并进一步分析了与发育相关的基因。共拼接得到49,436个unigenes并进行了注释和聚类，其中13,727个在NR数据库得到了注释，5,087个根据GO注释进行了分类，2,735个与189个KEGG通路有关。通过对不同胚胎发育时期的基因表达进行差异分析，共鉴定出6,658个差异表达基因。共有3,300个unigenes被鉴定为复眼色素形成期（EP）与孵化准备期（PH）之间的差异表达基因，其中1,595个被注释到数据库中；5,211个unigenes被鉴定为EP与幼体时期（L）之间的差异表达基因，其中2,540个被注释；1,262个unigenes被鉴定为PH与L之间的差异表达基因，其中680个被注释。本研究关注点主要是与形态或性状特征、信号通路及免疫系统相关的差异表达基因。与神经发生相关的Ato、Slit和Robo基因，与体分节相关的Cnc和mlpt基因及与眼睛发育相关的Apontic、Ey、Pax6和So基因均在EP期大量表达，说明EP期是神经系统形成、身体分节和复眼形成的关键时期；与附肢形成相关的Projectin基因在EP期和PH期表达量都比较高，表明EP期和PH期是附肢发育的主要阶段。Hedgehog、MAPK、Wnt、TGF-β和Notch等信号通路相关的发育基因在EP期比另两个时期的表达量高，表明EP比后两个时期具有更活跃的生物学过程。在三个胚胎发育时期，与过氧化物酶体、吞噬体和溶酶体相关的免疫基因丰富，说明红螯螯虾在胚胎时期已经形成了相对完整的免疫系统，并且吞噬细胞发挥主要的作用。

2、红螯螯虾胚胎发育过程中miRNA的鉴定与分析

运用miRNA组学鉴定了红螯螯虾三个胚胎发育时期（EP、PH和L）的miRNA及其靶基因。共获得19个已知miRNA和331个新miRNA，这些miRNA属于50个miRNA家族。三个胚胎发育时期两两比较，共鉴定出113个差异表达miRNA，预测出2,575个靶基因，其中1,257个被注释到各数据库中。此外，有9个miRNA及其63个靶基因被发现与胚胎发育相关。其中，miR-10及其靶基因可能调控神经系统发育和体分节。MiR-2788可能通过调节细胞增殖影响胚胎发育。还有miR-28（靶基因tutl）、miR-50（靶基因fbx5）和miR-1260b（靶基因sif）可能共同调控红螯螯虾胚胎眼睛的发育。分析了miRNA与其负调控的靶基因之间共同调控网络，这些miRNA及其靶基因构成的网络共同调控红螯螯虾胚胎组织和器官的发育。

3、红螯螯虾C型凝集素（CqCTL）的基因克隆及表达

根据组学部分结果研究克隆并鉴定了红螯螯虾第一个C型凝集素基因（CqCTL）。CqCTL的完整cDNA序列包含一个543 bp的开放阅读框，它编码的蛋白质含有180个氨基酸。对CqCTL的氨基酸序列分析表明，CqCTL中含有一个糖识别结构域（CRD），在CRD中含有4个保守半胱氨酸（Cys⁴⁸、Cys⁵⁹、Cys⁷⁶和Cys¹⁷⁷）以及决定结合特异性的EPD（Glu⁸⁰-Pro⁸¹-Asn⁸²）和QPD（Gln¹⁴⁶-Pro¹⁴⁷-Asn¹⁴⁸）基序。CqCTL与之前报道的其它物种C型凝集素具有高度的相似性。CqCTL的mRNA在所有十个组织中均可被检测到，且在肝胰腺中表达量最高。将编码CqCTL的cDNA片段重组到pET-32a(+)载体中，并在大肠杆菌BL21 (DE3) pLysS中表达。CqCTL以可溶性融合蛋白的形式表达，在其N端还包括Trx-、His-和S-标签。镍柱亲和层析法纯化蛋白，获得了表观分子量约为37 kDa的可溶性CqCTL。

4、红螯螯虾Kazal型丝氨酸蛋白酶抑制因子（CqKPI）的基因克隆、表达及功能研究

根据组学部分结果研究克隆并鉴定了红螯螯虾第一个Kazal型丝氨酸蛋白酶抑制因子（CqKPI）。CqKPI的开放阅读框包含405 bp，编码的蛋白质含有134个氨基酸序列。CqKPI具有两个Kazal结构域，均由44个氨基酸残基组成，具有保守氨基酸序列C-X₃-C-X₅-PVCG-X₅-Y-X₃-C-X₆-C-X₁₂-C，即每个Kazal结构域含有6个保守半胱氨酸，可以形成三对二硫键稳定Kazal结构域的构象。两个Kazal结构域P1位点的氨基酸残基分别为Ser⁴³和Lys⁹¹，表明CqKPI可能可以抑制胰蛋白酶和弹性蛋白酶的活性。CqKPI与之前报道的其它物种Kazal型丝氨酸蛋白酶抑制因子具有高度的相似性。CqKPI的mRNA在十个组织中均可被检测到，且在血细胞中表达量最高。重组的CqKPI在大肠杆菌中成功表达，并利用镍柱亲和层析柱和分子筛层析柱纯化，以供进一步研究。重组CqKPI可以与花津滩芽孢杆菌、金黄色葡萄球菌、副溶血弧菌和白色假丝酵母结合；还可抑制花津滩芽孢杆菌和白色假丝酵母的生长。这些结果表明CqKPI可能通过结合病原菌，与病原菌丝氨酸蛋白酶相互作用来参与红螯螯虾先天免疫系统，帮助红螯螯虾抵抗病原体的入侵。

The red claw crayfish (Cherax quadricanatus) is an emerging and important commercial aquatic animal and is also a potential biological model in crustacean biology due to its biological characteristics. Embryo development is the starting point of ontogeny and the quality of embryo development is one of the important factors affecting the constitution of crayfish. The pathogen is another important factor affecting crayfish culture. The innate immune system is a natural defense system for crustaceans to remove and kill pathogens. The research on embryo development and innate immunity of crayfish is of great significance to enrich the development pattern and immunity research of crustaceans, and also provides important guidance for disease prevention of crayfish culture. In the present study, the transcriptome and microRNA omics techniques were used to study the different embryonic development stages of C. quadricanatus. Meanwhile, based on the transcriptome data, gene cloning and functional verification of C-type lectin and Kazal-type serine protease inhibitor involved in innate immunity were conducted. The main research results are as follows:

1、A comparative transcriptomic analysis of C. quadricarinatus embryogenesis

High-throughput Illumina sequencing technology was used to investigate transcriptome profiles of three embryonic development stages of C. quadricarinatus and further analyzed genes related to development. A total of 49,436 unigenes were annotated and clustered, of which 13,727 were annotated in the NR database, 5,087 were classified according to GO annotation, and 2,735 were related to 189 KEGG pathways. Furthermore, a total of 6,658 differentially expressed genes (DEGs) were identified by differential analysis of gene expression among different embryonic development stages. A total of 3,300 unigenes were identified as DEGs between the eye pigments forming stage (EP) and prepare-hatching stage (PH), of which 1,595 were annotated to the database; 5,211 unigenes were identified as DEGs between EP and larvae (L), of which 2,540 were annotated; 1,262 unigenes were identified as DEGs between PH and L, of which 680 were annotated. This study focused on differentially expressed genes related to morphological or trait characteristics, signaling pathways and immune system. Ato, Slit and Robo genes related to neurogenesis, Cnc and mlpt genes related to body segmentation and Apontic, Ey, Pax6 and So genes related to eye development were all largely expressed in the EP stage, indicating that EP stage is a critical period for the formation of nervous system, body segmentation and eye. Projectin genes related to the formation of appendages were highly expressed in EP and PH phases, indicating that EP and PH phases were the main stages of appendage development. The signaling pathways such as Hedgehog, MAPK, Wnt, TGF- beta, and Notch, were higher expressed in the EP stage than in the other two stages, suggesting that EP has more active biological process than in the latter two stages. During the three embryonic development stages, immune genes related to peroxisome, phagocytic and lysosome were abundant, suggesting that C. quadricarinatus had formed a relatively complete immune system in the embryonic stage, and phagocytes played a major role.

2、Identification and profiling of microRNAs during embryogenesis in C. quadricarinatus

MiRNAs and their target genes were identified during three embryonic developmental stages of C. quadricarinatus. Nineteen known miRNAs and 331 novel ones belonging to 50 miRNA families were obtained. A total of 113 differentially expressed miRNAs were identified, and 2,575 target genes were predicted, among which 1,257 were annotated. Additionally, 63 target genes of 9 miRNAs in C. quadricarinatus were found to be related to embryonic development. For example, miR-10 and its target genes may regulate nervous system development and body segmentation. MiR-2788 may regulate cell proliferation to impact embryonic development. Moreover, miR-28 (target gene tutl), miR-50 (target gene fbx5) and miR-1260b (target gene sif) may co-regulate eye development of C. quadricarinatus embryonic. A regulatory network between miRNA and its negatively regulated target genes was analyzed. These miRNAs together with their target genes constitute a network for regulating the development of tissues and organs in the embryo of C. quadricarinatus.

3、Molecular cloning and expression of C-type lectin (CqCTL) from C. quadricarinatus

The first C. quadricarinatus C-type lectin gene (designated CqCTL) was cloned and characterized based on the results of transcriptome. The complete cDNA sequence of CqCTL contained an open reading frame (ORF) of 543 bp, which encoded a protein of 180 amino acids. A carbohydrate recognition domain (CRD) containing four conserved cysteines (Cys⁴⁸, Cys⁵⁹, Cys⁷⁶ and Cys¹⁷⁷) and the EPD (Glu⁸⁰-Pro⁸¹-Asn⁸²) and QPD (Gln¹⁴⁶-Pro¹⁴⁷-Asn¹⁴⁸) motifs were identified in the deduced amino acid sequence of CqCTL. CqCTL exhibited high similarity with previously identified C-type lectins from other species. The mRNA transcripts of CqCTL were ubiquitously detectable in all the tested ten tissues, with the highest expression level in hepatopancreas. The cDNA fragment encoding the mature peptide of CqCTL was recombined into pET-32a(+) with N-terminal Trx-, His-, and S- tags fused in-frame and expressed in Escherichia coli BL21 (DE3) pLysS, and an apparent MW of 37 kDa soluble CqCTL was obtained by using affinity chromatography method.

4、Molecular cloning, expression and functional study of Kazal-type serine protease inhibitor (CqKPI) from C. quadricarinatus

Kazal-type serine protease inhibitor (designated CqKPI) was cloned and identified based on the results of transcriptome in C. quadricarinatus. The ORF of CqKPI contained 405 nucleotides and encoded a protein of 134 amino acids. CqKPI had two Kazal domains, both of which were composed of 44 amino acid residues, with the conserved amino acid sequence C-X₃-C-X₅-PVCG-X₅-Y-X₃-C-X₆-C-X₁₂-C, that is, each Kazal domain contained 6 conserved cysteines, which could form the conformation of three pairs of disulfide bond stabled Kazal domain. The P1 amino acids of the Kazal domains were Ser⁴³ and Lys⁹¹, respectively, suggesting that CqKPI may inhibit trypsin and elastase. CqKPI exhibited high similarity with previously identified Kazal-type serine protease inhibitors from other species. The mRNA transcripts of CqKPI were ubiquitously detectable in all the tested ten tissues, with the highest expression level in hemocytes. The recombinant CqKPI was successfully expressed in E. coli and purified by Ni-NTA chromatography and size-exclusion chromatography. The recombinant CqKPI could bound to Bacillus hwajinpoensis, Staphylococcus aureus, Vibrio parahaemolyticus, and Candida albicans, and inhibit the growth of B. hwajinpoensis and C. albicans. These results indicate that CqKPI may participate in the innate immune system of C. quadricarinatus by binding pathogenic bacteria and interacting with serine protease of pathogens to help C. quadricarinatus resist the invasion of pathogens.

第1章  绪论.... 1

1.1  红螯螯虾胚胎发育及组学应用... 2

1.1.1  红螯螯虾胚胎发育研究进展... 2

1.1.1.1  形态学研究... 2

1.1.1.2  生化组成研究... 3

1.1.1.3  酶学研究... 4

1.1.1.4  环境影响... 4

1.1.1.5  分子生物学研究... 5

1.1.2  组学技术及其在甲壳动物胚胎发育研究中的应用前景... 6

1.1.2.1  组学技术的发展... 6

1.1.2.2  组学技术在甲壳动物胚胎研究中的应用... 7

1.2  甲壳动物先天免疫... 9

1.2.1  C型凝集素的研究进展... 9

1.2.1.1  模式识别受体... 9

1.2.1.2  C型凝集素的结构特点... 12

1.2.1.3  C型凝集素的生物学功能... 13

1.2.2  Kazal型丝氨酸蛋白酶抑制因子的研究进展... 15

1.2.2.1  丝氨酸蛋白酶和丝氨酸蛋白酶抑制因子... 15

1.2.2.2  Kazal型丝氨酸蛋白酶抑制因子的作用机理和抑制特异性... 16

1.2.2.3  Kazal型丝氨酸蛋白酶抑制因子的生物学功能... 18

1.3  本研究的目的和意义... 20

第2章  红螯螯虾胚胎发育转录组比较分析.... 21

2.1  引言... 21

2.2  材料与方法... 22

2.2.1  胚胎取样... 22

2.2.2  总RNA提取... 22

2.2.3  文库构建和Illuminate测序... 22

2.2.4  Reads质量控制、转录组组装与功能注释... 23

2.2.5  差异表达基因的鉴定... 23

2.2.6  荧光定量PCR验证... 24

2.3  结果... 25

2.3.1  胚胎发育时期的鉴定... 25

2.3.2  测序拼接与聚类... 26

2.3.3  功能基因注释与分类... 28

2.3.4  差异表达基因分析... 31

2.3.5  差异表达基因功能富集分析... 32

2.3.6  荧光定量PCR验证... 38

2.4  讨论... 40

2.5  小结... 43

第3章  红螯螯虾胚胎发育过程中microRNA的鉴定与分析.... 44

3.1  引言... 44

3.2  材料与方法... 44

3.2.1  胚胎取样与总RNA的提取... 45

3.2.2  小RNA文库构建与测序... 45

3.2.3  红螯螯虾miRNA的鉴定... 45

3.2.4  miRNA的差异表达分析... 46

3.2.5  miRNA靶基因的预测... 46

3.2.6  miRNA及靶基因共表达分析... 46

3.2.7  荧光定量PCR验证miRNA.. 46

3.3  结果... 47

3.3.1  小RNA高通量测序... 47

3.3.2  miRNA的鉴定... 48

3.3.3  差异表达miRNA的鉴定... 50

3.3.4  差异表达miRNA靶基因的预测及注释... 52

3.3.5  差异表达miRNA关联靶基因共表达分析... 57

3.3.6  荧光定量PCR验证miRNA.. 57

3.4  讨论... 60

3.5  小结... 62

第4章  红螯螯虾C型凝集素（CqCTL）的基因克隆及表达.... 63

4.1  引言... 63

4.2  材料与方法... 64

4.2.1  实验材料... 64

4.2.1.1  实验动物、菌株和质粒... 64

4.2.1.2  实验仪器和设备... 65

4.2.1.3  实验试剂和耗材... 65

4.2.1.4  实验试剂配制... 67

4.2.2  实验方法... 68

4.2.2.1  总RNA提取和cDNA合成... 68

4.2.2.2  CqCTL的克隆... 68

4.2.2.3  CqCTL的生物信息学分析... 70

4.2.2.4  CqCTL mRNA表达的定量分析... 71

4.2.2.5  CqCTL的重组表达及纯化... 71

4.3  结果... 72

4.3.1  CqCTL cDNA克隆与鉴定... 72

4.3.2  CqCTL的序列比对与进化树分析... 74

4.3.3  CqCTL mRNA的组织分布... 77

4.3.4  重组CqCTL的表达和纯化... 78

4.4  讨论... 79

4.5  小结... 81

第5章  红螯螯虾Kazal型丝氨酸蛋白酶抑制因子（CqKPI）的基因克隆、表达及功能研究.... 82

5.1  引言... 82

5.2  材料与方法... 83

5.2.1  实验材料... 83

5.2.2  实验方法... 83

5.2.2.1  总RNA提取和cDNA合成... 83

5.2.2.2  CqKPI的克隆... 83

5.2.2.3  CqKPI的生物信息学分析... 84

5.2.2.4  CqKPI mRNA表达的定量分析... 84

5.2.2.5  CqKPI的重组表达及纯化... 84

5.2.2.6  重组CqKPI微生物结合实验... 85

5.2.2.7  Western blot法检测蛋白... 85

5.2.2.8  微生物生长抑制实验... 86

5.3  结果... 86

5.3.1  CqKPI cDNA克隆与鉴定... 86

5.3.2  CqKPI的序列比对与进化树分析... 87

5.3.3  CqKPI mRNA的组织分布... 90

5.3.4  重组CqKPI的表达和纯化... 91

5.3.5  重组CqKPI微生物结合活性... 92

5.3.6  重组CqKPI微生物生长抑制活性... 93

5.4  讨论... 94

5.5  小结... 95

第6章  结论、创新点与展望.... 97

6.1  主要结论... 97

6.2  创新点... 98

6.3  展望... 98

参考文献.... 99

致  谢.... 119

作者简历及攻读学位期间发表的学术论文与研究成果.... 121