学校编码 :10384 分类号密级学号 : UDC 硕士学位论文赤点石斑鱼 MHC 基因的克隆与多态性分析 The cloning and polymorphism of MHC gene from Epinephelus akaara 杜佳莹指导教师姓名 : 王

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学校编码 :10384 分类号密级学号 :21120051302182 UDC 硕士学位论文赤点石斑鱼 MHC 基因的克隆与多态性分析 The cloning and polymorphism of MHC gene from Epinephelus akaara

1 学校编码 :10384 分类号密级学号 : UDC 硕士学位论文赤点石斑鱼 MHC 基因的克隆与多态性分析 The cloning and polymorphism of MHC gene from Epinephelus akaara 杜佳莹指导教师姓名 : 王军教授专业名称 : 海洋生物学论文提交日期 :2008 年 5 月论文答辩时间 :2008 年 6 月学位授予日期 : 年月答辩委员会主席 : 陈奕欣教授评阅人 : 陈奕欣教授王克坚教授林祥志副研究员 2008 年 6 月

2 厦门大学学位论文原创性声明兹呈交的学位论文, 是本人在导师的指导下独立完成的研究成果本人在论文写作过程中参考其他个人或集体的研究成果, 均在文中以明确方式标明本人依法享有和承担由此论文而产生的权利和责任声明人 ( 签名 ): 年月日

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5 摘要摘要主要组织相容性复合体 (Major histocompatibility complex, MHC) 是免疫系统中对蛋白抗原异物起反应的一类重要的免疫因子, 广泛分布于各种脊椎动物体内本研究以我国具有重要经济价值和养殖前景的赤点石斑鱼 (Epinephelus akaara) 为实验材料, 采用 PCR RT-PCR SMART-RACE SSCP 等技术对 MHC 家族 MHC IA MHC IIA 和 MHC IIB 3 个基因进行克隆与多态性分析主要结果如下 : 1. 本研究首次成功克隆获得赤点石斑鱼的 MHC class Iα 链,MHC class II α 链及 β 链等 3 个重要的非特异性免疫因子的 cdna 全序列, 其中 MHC class I α 链全长 1720bp, 含 3 UTR 启动子多肽结合区(α 1 和 α 2 ) 免疫球蛋白样区(α 3 ) 跨膜区胞内区和 5 UTR MHC class II α 链全长 1870bp, 含 3 UTR 启动子多肽结合区 (α 1 ) 免疫球蛋白样区(α 2 ) 跨膜区胞内区和 5 UTR MHC class II β 链全长 1350bp, 含 3 UTR 启动子多肽结合区(β 1 ) 免疫球蛋白样区(β 2 ) 跨膜区胞内区和 5 UTR 2. 3 个免疫蛋白因子均具有经典 MHC 蛋白分子的空间结构, 在多肽结合区均存在极其丰富的变异其中 Iα 链的 α 1 (1-90 位 ) 和 α 2 ( 位 ) 分别由 1 个 α 螺旋和 4 个反向平行的 β 折叠组成, 形成 1 个深沟状凹槽,α 3 ( 位 ) 主要由 β 折叠组成 IIα 链的 α 1 (24~99 位 ) 由 1 个 α 螺旋和 4 个反向平行的 β 折叠组成,α 2 (100~200 位 ) 由多个 β 折叠组成 IIβ 链的 β 1 (20~103 位 ) 1 个 α 螺旋与 4 个反向平行的 β 折叠构成多肽结合区,β 2 (116~204 位 ) 由多个 β 折叠构成两个反向平行的三明治结构 3. 3 个非特异性免疫因子在脑心脾肝头肾肠肌肉血细胞胸腺性腺, 鳃, 胃等 12 个组织均能有效表达, 在头肾, 脾, 胸腺等免疫组织表达量不仅高, 而且表达多态性也异常丰富递呈内源性抗原的 MHC class I 在与外界病原接触较多的鳃, 肌肉, 肠等组织表达较弱, 而递呈外源性抗原的 MHC class II 在鳃, 肌肉, 肠等组织则有较强表达, 体现了其表达与功能的适应性 4. 建立了利用 SSCP 技术分离多肽 MHC 基因片段的方法, 极大提高了 MHC 多态性基因的检测效率本研究所获得的 SSCP 特异性引物及其建立的检 I

6 摘要测方法, 将可为今后大量检测赤点石斑鱼多态性 MHC 序列, 筛选可针对特定病原的特异序列提供有力的工具 5. 根据 3 个分子的 IGC 区氨基酸序列构建了脊椎动物的系统进化树, 其拓扑结构基本一致, 不仅证实了硬骨鱼类的单系起源, 而且支持了 MHC 系统分化早于原始鱼类向更高等脊椎动物进化的观点, 同时也表明了 3 个分子的 IGC 区氨基酸序列可以作为研究鱼类物种间进化关系的良好标记关键词 :MHC 基因 ; 赤点石斑鱼 ; 多态性 II

7 Abstactr Abstract The major histocompatibility complex molecules (MHC I and MHC II) are fundamental components of imumune response to foreign protein antigens, and occur extensively in all vertebrate organisms. In this study, using molecular techniques such as PCR, RT-PCR,SMART-RACE, SSCP, we report the cloning and polymorphism of three MHC genes from Epinephelus akaara; one of the important aquaculture species in China. The results are shown as follows: 1. It was the first time that the complete cdna sequences of three important non-specific immune genes, MHC class I α chain, MHC class II α chain and MHC class II β chain were obtained from the grouper. The whole MHC class I α chain cdna sequence has 1720bp, which consists of: a 3 UTR (untranscribed region), promoters, a peptide-binding region (α1 and α2), an immunoglobulin-like region (α3), a transmembrane region, a cytoplasmic region and a 5 UTR. The MHC class II α chain has 1870bp, which consists of: a 3 UTR, promoters, a peptide-binding region (α1), an immunoglobulin-like region (α2), a transmembrane region, a cytoplasmic region and a 5 UTR. Likewise, the MHC class II β chain has 1350bp, which consists of: a 3 UTR, promoters, a peptide-binding region (β 1 ), an immunoglobulin-like region (β 2 ), a transmembrane region, a cytoplasmic region and a 5 UTR. 2. The tesed three genes have classic MHC 3D molecular structure, and exhibit abundant polymorphisms in the peptide-binding region. Both α1 and α2 domains of the class I α chain consist of an alpha-helical region and a beta sheet of four strands in antiparallel orientation, forming a deep gorge, while the α 3 domain consists of several beta strands. Both α 1 and β 1 of MHC II were composed of an alpha-helical region and a beta sheet of four strands in antiparallel orientation, while α 2 and β 2 of MHC II form a sandwich-like structure made of two antiparallel sheets, each consisting of several strands. 3. The above 3 MHC genes are all expressed in brain, heart, spleen, liver, III

8 Abstract head-kidney, intestines, muscle, blood, thymus, gonad, gill and stomach, and higher levels of expression and polymorphism were observed in immune organs such as head-kidney, spleen and thymus. However, in organs such as gills and muscles that more likely to be frequently exposed to bacterial antigens, the expression and polymorphism levels are lower in MHC I while still elevated in MHC II. This pattern demonstrates the adaptability of MHC molecules in both expression and function. 4. Using the SSCP technique, we established a protocol to separate the peptidebinding domain of MHC genes. By this protocol, one can enhance the detection efficiency of the MHC gene polymorphisms enormously. The SSCP primers and the protocol developed in this research will be helpful for the future study in detecting polymorphic sequences of MHC in groupers including E. akaara. 5. Phylogenetic trees were also constructed based on the Neighbour-Joining (NJ) methods with the IGC region of these three genes. Similar tree topologies for all three genes confirm the monophyly of the Teleosteans, and also demonstrates that MHC-I and MHC-II diverged before ancestral fish gave rise to higher vertebrates. Therefore, the amino acid sequences of the IGC region of these three genes are potentially informative for phylogenetic studies in fishes. Key words : Major histocompatibility complex; Epinephelus akaara; polymorphism IV

9 目录目录中文摘要 I 英文摘要 III 第一章绪言鱼类 MHC 基因及其研究进展鱼类的免疫系统 MHC 基因简介 MHC 基因的生物学特性 MHC 基因家族 MHC 基因的主要功能鱼类 MHC 基因的分布与表达特性 MHC 基因在鱼类研究中的应用与展望赤点石斑鱼研究进展赤点石斑鱼的形态特征赤点石斑鱼的地理分布当前赤点石斑鱼的相关研究本研究的目的与意义...16 第二章赤点石斑鱼 MHC IA 基因的克隆和多态性分析材料样品的采集与鉴定主要仪器主要试剂常用溶液配制方法 mrna 的提取 RT-PCR 扩增 PCR 引物设计 SMART-RACE 扩增琼脂糖凝胶电泳 PCR 产物的回收和纯化序列测定 SSCP 分析序列拼接及 cdna 全序列分析结果赤点石斑鱼各组织总 RNA 的提取 MHC IA 基因全长 cdna 的克隆和序列分析...31

10 目录 3.3 MHC IA 基因的氨基酸序列分析和蛋白质二级结构和空间结构预测赤点石斑鱼 MHC IA 基因的组织表达多态性分析错误! 未定义书签 4 讨论赤点石斑鱼 MHC IA 基因的序列特征分析赤点石斑鱼 MHC IA 基因的空间结构分析赤点石斑鱼 MHC IA 基因的表达多态性分析 MHC IA 基因的同源性和系统发生分析...41 第三章赤点石斑鱼 MHC IIA 基因的克隆和多态性分析材料方法 mrna 的提取 RT-PCR 扩增 PCR 引物设计 SMART-RACE 扩增琼脂糖凝胶电泳 PCR 产物的回收和纯化序列测定 SSCP 分析序列拼接及 cdna 全序列分析结果 MHC IIA 基因全长 cdna 的克隆和序列分析 MHC IIA 基因的氨基酸序列分析和蛋白质二级结构和空间结构预测赤点石斑鱼 MHC IIA 基因的组织表达多态性分析错误! 未定义书签 4 讨论赤点石斑鱼 MHC IIA 基因的序列特征分析赤点石斑鱼 MHC IIA 基因的空间结构分析赤点石斑鱼 MHC IIA 基因的表达多态性分析 MHC IIA 基因的同源性和系统发生分析...62 第四章赤点石斑鱼 MHC IIB 基因的克隆和多态性分析材料方法 RT-PCR 扩增 PCR 引物设计 SMART-RACE 扩增琼脂糖凝胶电泳 PCR 产物的回收和纯化序列测定 SSCP 分析序列拼接及 cdna 全序列分析结果...72

11 目录 3.1 MHC IIB 基因全长 cdna 的克隆和序列分析 MHC IIB 基因的氨基酸序列分析和蛋白质二级结构和空间结构预测赤点石斑鱼 MHC IIB 基因的组织表达多态性分析错误! 未定义书签 4 讨论赤点石斑鱼 MHC IIB 基因的序列特征分析赤点石斑鱼 MHC IIB 基因的空间结构分析赤点石斑鱼 MHC IIB 基因的表达多态性分析 MHC IIB 基因的同源性和系统发生分析...81 结论...86 附图...87 参考文献...90 在学期间发表论文...99 致谢语...100

13 CONTENTS CONTENTS Abstract in Chinese..I Abstract in English III Chapter 1 Introduction The Progress of MHC Gene Researches in Fish The immunity system of fish Brief introduction of MHC gene Biological characteristics of MHC gene The family of MHC gene Primary function of MHC gene The distribution and expressive characteristics Application and prospect of MHC gene research in fish Introduction of the researching objects The morphologic characteristics of Epinephelus akaara The geographical distribution of Epinephelus akaara Recently research in Epinephelus akaara Purpose and significance of the research...17 Chapter 2 The cloning and polymorphism of MHC IA gene of Epinephelus akaara Materials Sample collection and identification Main apparatus Main reagent Confection of solution in common use Methods Methods of RNA extraction RT-PCR amplification Primers design SMART-RACE amplification Agar gel Electrophoresis Purification of PCR products Sequencing SSCP Electrophoresis Data analysis Results Results of RNA extraction of E.akaara...31

14 CONTENTS 3.2 The cloning and analysis of MHC IA gene from E.akaara The amino acid sequence and protein structure of MHC IA gene The polymorphism of organic expression of MHC IA gene Discussion Sequence characteristics analysis of MHC IA gene The tridimensional structure analysis of MHC IA gene The expressive polymorphism analysis of MHC IA gene The Phylogenetic evolution analysis of MHC IA gene...41 Chapter 3 The cloning and polymorphism of MHC IIA gene from Epinephelus akaara Materials Methods Methods of RNA extraction RT-PCR amplification Primers design SMART-RACE amplification Agar gel Electrophoresis Purification of PCR products Sequencing SSCP Electrophoresis Data analysis Results The cloning and analysis of MHC IIA gene from E.akaara The amino acid sequence and protein structure of MHC IIA gene The polymorphism of organic expression of MHC IIA gene Discussion Sequence characteristics analysis of MHC IIA gene The tridimensional structure analysis of MHC IIA gene The expressive polymorphism analysis of MHC IIA gene The Phylogenetic evolution analysis of MHC IIA gene...62 Chapter 4 The cloning and polymorphism of MHC IIB gene from Epinephelus akaara Materials Methods RT-PCR amplification Primers design SMART-RACE amplification...67

15 CONTENTS 2.4 Agar gel Electrophoresis Purification of PCR products Sequencing SSCP Electrophoresis Data analysis Results The cloning and analysis of MHC IIB gene from E.akaara The amino acid sequence and protein structure of MHC IIB gene The polymorphism of organic expression of MHC IIB gene Discussion Sequence characteristics analysis of MHC IIB gene The tridimensional structure analysis of MHC IIB gene The expressive polymorphism analysis of MHC IIB gene The Phylogenetic evolution analysis of MHC IIB gene...81 Conclusion...86 Attached Drawing...87 References...90 Publications...99 Acknowledgements...100

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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学校编码 :10384 分类号密级 学号 : UDC 硕士学位论文 赤点石斑鱼 MHC 基因的克隆与多态性分析 The cloning and polymorphism of MHC gene from Epinephelus akaara 杜佳莹 指导教师姓名 : 王

学校编码 :10384 分类号密级学号 : UDC 硕士学位论文赤点石斑鱼 MHC 基因的克隆与多态性分析 The cloning and polymorphism of MHC gene from Epinephelus akaara 杜佳莹指导教师姓名 : 王