HUAZHONG AGRICULTURAL UNIVERSITY 博 士 学 位 论 文 PhD DISSERTATION TRANSCRIPTOME ANALYSIS AND EXPRESSION OF IMMUNE-RELATED GENES IN MEGALOBRAMA AMBLYCEPHALA AFTER CHALLENGE WITH AEROMONAS HYDROPHILA 研 究 生: AS REVEALED BYTRAN MOLECULAR MARKERS CANDIDATE: NGOC TUAN 导 师: SUPERVISOR: 专 PROFESSOR WANG WEIMIN 业: MAJOR: AQUACULTURE 研究方向: RESEARCH AREA: FISH GENETICS AND BREEDING 中国 武汉 WUHAN，CHINA 2015 年 月 JUNE，2015 分类号 密级 Huazhong Agricultural University Transcriptome Analysis and Expression of Immune-Related Genes in Megalobrama amblycephala after Challenge with Aeromonas hydrophila Name: Tran Ngoc Tuan Advisor: Professor Wang Weimin Major: Aquaculture Subject: Fish Genetics and Breeding Degree: Doctor of Philosophy Date of receiving certificate: June 2015 College of Fisheries Huazhong Agricultural University June 2015 Transcriptome analysis and expression of immune-related genes in M amblycephala after challenge with A hydrophila 华中农业大学学位论文独创性声明及使用授权书 I 华中农业大学 2015 届博士研究生学位论文 CONTENTS 摘 要 ABSTRACT ABRBREVIATIONS CHAPTER Review of literature 1.1 General introduction 1.2 Introduction to blunt snout bream 1.2.1 Biological characteristics 1.2.2 Interests to research 10 1.3 Aeromonas hydrophila and its infection in fish 16 1.3.1 Classification and identification 16 1.3.2 Pathology 17 1.3.3 Clinical signs 17 1.3.4 Virulence factors 18 1.3.5 Infection of Aeromonas to blunt snout bream 18 1.4 Transcriptome study 19 CHAPTER Pathogenicity of Aeromonas hydrophila to Megalobrama amblycephala 23 2.1 Introduction 23 2.2 Materials and Methods 23 2.2.1 Bacterial strain and bacterial suspension preparation 23 2.2.2 Fish for experiment 24 2.2.3 Pathogenicity test 24 2.2.4 Biochemical tests 25 2.2.5 Antibiotic susceptibility test 25 2.2.6 Molecular analysis 26 2.3 Results 26 2.3.1 Clinical signs 26 2.3.2 The cumulative mortality 27 2.3.3 Re-isolation and re-identification 27 2.3.4 16S rRNA sequences analysis 29 II Transcriptome analysis and expression of immune-related genes in M amblycephala after challenge with A hydrophila 2.3.5 Antimicrobial susceptibility test 29 2.4 Discussion 30 CHAPTER Transcriptome analysis of the Megalobrama amblycephala after challenge with Aeromonas hydrophila 34 3.1 Introduction 34 3.2 Materials and Methods 35 3.2.1 Fish and bacterial challenge 35 3.2.2 Sampling and RNA extraction 35 3.2.3 cDNA library preparation and Illumina sequencing 36 3.2.4 Sequence quality assessment and de novo assembly 37 3.2.5 Annotation 37 3.2.6 Differential gene expression analysis 37 3.2.7 Identification of immune-related genes 38 3.3 Results 38 3.3.1 Transcriptome sequencing and assembly 38 3.3.2 BLAST search analysis 40 3.3.3 Unigene functional annotation 41 3.3.4 Analysis of differentially expressed gene profiles 45 3.3.5 Identification of differentially expressed innate immune-related genes 46 3.3.6 Pathway analysis 47 3.4 Discussion 50 CHAPTER Simple sequence repeat and single nucleotide polymorphism markers identified from Megalobrama amblycephala transcriptome profile 54 4.1 Introduction 54 4.2 Materials and Methods 55 4.2.1 SSR markers 55 4.2.2 SNP markers 55 4.3 Results 56 4.3.1 SSR markers 56 4.3.2 SNP markers 57 III 华中农业大学 2015 届博士研究生学位论文 4.4 Discussion 63 CHAPTER In silico characterization and homology modeling of proteins encoded by immune-related genes in Megalobrama amblycephala 65 5.1 Introduction 65 5.2 Materials and Methods 66 5.2.1 Sequence identification 66 5.2.2 Bioinformatics analysis 66 5.2.3 Protein structure prediction 67 5.3 Results 67 5.3.1 Sequence analysis 67 5.3.2 Physicochemical and functional characterization analysis 106 5.3.2 Protein structure prediction 110 5.4 Discussion 118 CHAPTER Expression of immune-related genes in the Megalobrama amblycephala after Aeromonas hydrophila infection 124 6.1 Introduction 124 6.2 Materials and Methods 126 6.2.1Transcripts source 126 6.2.2 Fish and challenge experiment 126 6.2.3 Total RNA preparation and cDNA synthesis 126 6.2.4 Quantitative real-time PCR (qPCR) and statistics 127 6.3 Results 127 6.3.1 MaTLR5 128 6.3.2 MaNFKBIA 128 6.3.3 MaMyD88 129 6.3.4 MaTRAF6 129 6.3.5 MaC3 129 6.3.6 MaC7 130 6.3.7 MaMMP-9 130 6.3.8 MaCTSL 130 6.3.9 MaIL-8 130 IV Transcriptome analysis and expression of immune-related genes in M amblycephala after challenge with A hydrophila 6.3.10 MaIL-10 131 6.4 Discussion 133 6.4.1 Genes related to TLR pathway 134 6.4.2 Genes related to complement and coagulation cascades 135 6.4.3 Genes related to antigen processing and presentation and leukocyte transendothelial migration 136 6.4.4 Genes related to cytokine-cytokine receptor interaction 138 CHAPTER Conclusion 140 REFERENCES 142 APPENDIX Transcriptome profile database of Megalobrama amblycephala by using Solexa/Illumina technology 172 APPENDIX Publications 173 APPENDIX Awards and Funding 175 ACKNOWLEDMENTS 176 V Transcriptome analysis and expression of immune-related genes in M amblycephala after challenge with A hydrophila 摘 要 团头鲂（Megalobrama amblycephala）是中国重要的淡水养殖草食性经济鱼类。 嗜水气单胞菌在团头鲂高密度养殖中的危害较为严重，可造成严重的经济损失。迄 今为止，有关团头鲂感染嗜水气单胞菌的分子调控机理还未见报道，本研究通过注 射嗜水气单胞菌对团头鲂进行攻毒，采用高通量测序技术对攻毒前后 个组织进行 了转录组分析及免疫基因的鉴定，分析了这些基因的调控与表达，探讨了团头鲂对 外源性病原体侵入的抵抗力和敏感性。主要内容如下： （1）对团头鲂腹腔注射 1.7×105，1.7×106，1.7×107 和 1.7×108 细胞每毫升等不 同浓度的菌液，并在七天的实验期观察和记录鱼的患病症状和死亡情况。试验结果 表明，试验鱼的死亡率与细菌的感染浓度相关。与对照组 0%相比，高浓度（1.7×108 细胞每毫升）表现出 100%死亡率（