实验海洋生物学重点实验室知识库

海豚链球菌（Streptococcus iniae, S. iniae）是海水养殖鱼类重要的细菌性病原，给水产养殖业造成了巨大的经济损失，严重阻碍了鱼类养殖业的健康发展。目前，愈来愈多的国内外研究人员从宿主（海水鱼类）免疫防御机制等方面开展系统的研究，以此为海豚链球菌病的防御提供理论基础和科学依据。

MicroRNA（miRNA）参与许多生物活动，包括对病原体的免疫防御。在本论文研究中，我们应用高通量测序技术分析海豚链球菌感染后不同时间点（6h和24h）牙鲆体内的miRNA表达情况。共鉴定出1038个miRNAs，其中249个miRNAs是新颖的miRNAs，81个miRNAs在感染海豚链球菌后呈现显著差异表达，被命名为DEmiRNAs（Differentially Expressed miRNAs）。在这81个DEmiRNAs中，34个和58个miRNA分别出现在感染后6小时和24小时。大多数DEmiRNAs具有很强的时间特异性，仅有13.6％的DEmiRNAs同时出现在两个时间点。对81个DEmiRNAs进行靶基因预测分析，发现了9582个预测靶基因。通过GO（Gene Ontology）分析，这些靶基因被富集到各种GO terms，主要包括生物过程（biological process）、分子功能（molecular function）和细胞组分（cellular component）。 KEGG （Kyoto Encyclopedia of Genes and Genomes）分析表明，这些靶基因也被富集到各种KEGG 通路，主要包括内吞作用和溶酶体相关途径以及MAPK、ErbB和AMPK等信号传导途径。

自上述发现的DEmiRNAs中，选择了两个DEmiRNAs ，即pol-3p-10740_175和pol-miR-216b，对其进行了调控与功能研究。发现pol-3p-10740_175靶向双重特异性磷酸酶6（dual specificity phosphatase 6，Dusp6），并抑制后者的表达。用pol-3p-10740_175转染牙鲆FG细胞可显著性抑制海豚链球菌入侵。发现pol-miR-216b的表达水平在海豚链球菌和细胞肿大病毒（megalocytivirus）感染过程中发生显著变化。进一步研究发现pol-miR-216b靶向Fas凋亡抑制分子（Fas apoptotic inhibitory molecule, transcript variant X1，FAIM1）。pol-miR-216b通过与牙鲆FAIM1（命名为PoFAIM1）的3’-UTR相互作用而调控PoFAIM1表达。Fas凋亡抑制分子是一种抗凋亡蛋白，其在鱼类中的功能作用尚不清楚。我们发现PoFAIM1与哺乳动物FAIMs在结构上高度保守，都有一个FAIM1结构域。为了研究PoFAIM1的功能，我们构建了干扰PoFAIM1表达的RNAi干扰表达质粒，并检测了它们的体内效应，发现干扰表达质粒能显著抑制PoFAIM1在牙鲆肝、脾、肾、肠中的表达。

综上所述，本论文研究首次鉴定了海豚链球菌诱导的牙鲆miRNA表达谱图，发现了海豚链球菌调控的大量miRNAs，证明了miRNA参与抗海豚链球菌感染免疫。这些研究结果加深了我们对鱼类miRNA在抗细菌感染免疫中的作用的理解。

Streptococcus iniae is a serious bacterial pathogen in marine fish. The outbreak of S. iniae disease has brought huge economic losses and severely hampered the development of fish farming industry. Now, more and more researchers have been studying the immune response mechanism of marine fish, which will provide theoretical and scientific basis for the prevention and treatment of bacterial disease in fish.

MicroRNAs (miRNAs) are involved in many biological activities including immune defense against pathogens. In this study, we applied high-throughput sequencing technology to examine miRNAs in Japanese flounder (Paralichthys olivaceus) infected with S. iniae at different times. A total of 1038 miRNAs were identified, of which, 249 were novel miRNAs, and 81 showed differential expression (named DEmiRNAs) after S. iniae infection. Of the 81 DEmiRNAs identified, 34 and 58 occurred at 6 h and 24 h post-infection, respectively; most DEmiRNAs were strongly time-specific, and only 13.6% of the DEmiRNAs were shared between the two time points. A total of 9582 target genes were predicted for the 81 DEmiRNAs. The putative target genes were enriched in various GO and KEGG pathways of biological processes and molecular/cellular functions, in particular endocytosis, regulation of transcription, lysososme, and the signaling pathways of MAPK, ErbB, and AMPK.

Two of the DEmiRNAs, pol-3p-10740_175 and pol-miR-216b were chosen for further study. pol-3p-10740_175 was found to target dual specificity phosphatase 6 (Dusp6) and repress the expression of Dusp6. Transfection of flounder FG cells with pol-3p-10740_175 caused a significant inhibition on S. iniae invasion. qRT-PCR analysis showed that pol-miR-216b expression was regulated by S. iniae and megalocytivirus in a pathogen-specific manner, and that pol-miR-216b targeted the gene of FAIM1 (Fas apoptotic inhibitory molecule, transcript variant X1，FAIM1) of flounder (named PoFAIM1) by negative interaction with the 3’-UTR of FAIM1. Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. However, its working mechanism and role in fish are unknown.We found that PoFAIM1 is highly conserved with mammalian FAIMs in structure and has a FAIM1 domain. To investigate the function of PoFAIM1, we constructed RNA interfering (RNAi) plasmids that expresses a small interfering RNA targeting PoFAIM1. Administration of flounder with the RNAi plasmids caused a significant inhibition on the expression level of PoFAIM1 in the liver, spleen, kidney and intestine, indicating that these plasmids can be used to knockdown the expression of PoFAIM1 in vivo.

The results of this study provided the first S. iniae-induced miRNA profile in Japanese flounder, identified a large number of S. iniae-induced host miRNAs and demonstrated that flounder miRNAs play an important role in antibacterial immunity. These results added new insights into the function of fish miRNAs in the immune defense against bacterial infection.

目 录

第1章 引言... 1

1.1 MicroRNA研究进展... 1

1.1.1 MiRNA的发现... 1

1.1.2 MiRNA的合成途径... 1

1.1.3 MiRNA的作用机制... 2

1.1.4 宿主miRNA介导的免疫防御和对微生物感染进程的影响... 4

1.1.5 MiRNA的临床应用... 5

1.1.6 MiRNA在水产病害中的研究进展... 6

1.2 海豚链球菌研究进展... 7

1.2.1 海豚链球菌的病原学基本特征... 7

1.2.2 海豚链球菌的地域分布... 7

1.2.3 海豚链球菌的宿主范围... 8

1.2.4 海豚链球菌感染的防控... 9

1.3双重特异性磷酸酶6（dual-specificity phosphatases 6，Dusp6）的研究进展... 10

1.3.1 MAPK信号通路... 10

1.3.2 Dusp6与MAPKs. 10

1.3.3 Dusp6与疾病治疗... 11

1.3.4 Dusp6在水生生物中的研究进展... 11

1.4 Fas凋亡抑制分子（Fas apoptotic inhibitory molecule, FAIM）研究进展... 12

1.4.1 细胞凋亡... 12

1.4.2 FAIM... 14

1.4.3 FAIM与疾病治疗... 15

1.5 本研究的目的与意义... 16

第2章 海豚链球菌感染条件下牙鲆miRNA表达谱的鉴定... 17

2.1 材料与方法... 18

2.1.1 实验材料... 18

2.1.2 感染实验和样品采集... 18

2.1.3 MiRNA文库建立和高通量测序... 19

2.1.4 MiRNA测序数据分析... 19

2.1.5 MiRNA靶基因预测... 20

2.1.6 反转录荧光定量PCR（qRT-PCR）... 20

2.2 结果... 23

2.2.1 感染和未感染海豚链球菌牙鲆的miRNAs鉴定... 23

2.2.2 海豚链球菌感染条件下牙鲆miRNAs的差异表达... 23

2.2.3 差异表达miRNAs的qRT-PCR验证... 25

2.2.4 DEmiRNAs靶基因预测和富集性分析... 26

2.3讨论和结论... 36

第3章 pol-3p-10740_175对靶基因的调控及对海豚链球菌侵染的影响... 39

3.1 材料与方法... 39

3.1.1 实验材料... 39

3.1.2 感染实验... 39

3.1.3 qRT-PCR.. 40

3.1.4 MiRNA模拟物（mimic）... 42

3.1.5 双荧光素酶报告基因检测... 42

3.1.6 海豚链球菌的细胞感染实验... 49

3.2 结果... 50

3.2.1 海豚链球菌感染对pol-3p-10740_175预测靶基因表达的影响... 50

3.2.2 pol-3p-10740_175 对Dusp6的调控作用... 51

3.2.3 pol-3p-10740_175对海豚链球菌感染的影响... 52

3.3 讨论和结论... 53

第4章 pol-miR-216b的靶基因鉴定及分析... 57

4.1 材料与方法... 57

4.1.1 实验材料... 57

4.1.2 感染实验... 58

4.1.3 qRT-PCR.. 58

4.1.4 双荧光素酶报告基因检测... 61

4.1.5 PoFAIM1的序列分析... 61

4.1.6 PoFAIM1的RNA干扰... 61

4.2 结果... 67

4.2.1 海豚链球菌和细胞肿大病毒感染对牙鲆pol-miR-216b和PoFAIM1表达的影响... 68

4.2.2 pol-miR-216b与PoFAIM1 3’UTR的相互作用... 69

4.2.3 PoFAIM1序列分析... 71

4.2.4 PoFAIM1体内表达干扰... 73

4.3 讨论和结论... 74

第5章 结论... 77

参考文献... 79

附录一 实验用溶液配制方法... 99

附录二 实验用仪器... 100

致 谢... 101

作者简历及攻读学位期间发表的学术论文与研究成果... 103