实验海洋生物学重点实验室知识库

    牙鲆（Paralichthys olivaceus）是我国重要的水产养殖鱼类，易受多种鱼类病原体包括迟缓爱德华氏菌（Edwardsiella tarda，E. tarda）的感染。E. tarda是一种宿主范围广泛的革兰氏阴性菌。MicroRNAs（miRNAs）是一类长度为21-23个核苷酸的单链非编码RNA，在转录后水平通过负调控靶基因的表达，参与多个细胞生物学过程的调节，包括病原感染、细胞自噬和细胞凋亡。在本论文研究中，我们采用高通量测序技术，研究了E. tarda在感染牙鲆后不同时间点，牙鲆肾脏miRNAs的表达情况。共有96个牙鲆miRNAs的表达在感染E. tarda后显示显著性差异，并被命名为DEmiRNAs（Differentially Expressed miRNAs）。针对这96个DEmiRNAs进行了靶基因预测分析，共发现2779个潜在靶基因。靶基因的富集分析显示，这2779个预测靶基因与多种生物过程、细胞组分和分子功能相关，并调控多条涉及免疫的通路。

    从上述96个DEmiRNAs中，我们挑选了四个差异倍数大或表达量丰富的DEmiRNAs，分别为pol-miR-182-5p、pol-miR-novel_171、pol-miR-novel_547和pol-miR-novel_395，研究了其对靶基因的调控机制以及在E. tarda感染过程中的调控功能。

        1、对于pol-miR-182-5p，我们发现其靶基因是S1PR1（sphingosine-1-phosphate receptor 1）。pol-miR-182-5p通过与牙鲆S1PR1（PoS1PR1）的3′UTR之间的特异性结合负调控PoS1PR1。过量表达pol-miR-182-5p显著促进牙鲆细胞凋亡，抑制细胞增殖。在牙鲆体内干扰PoS1PR1表达能增强E. tarda在鱼体组织中的侵袭和传播。

        2、对于pol-miR-novel_171，我们发现其表达受E. tarda和细胞肿大病毒（megalocytivirus）RBIV-C1感染的影响。研究发现FAM49B（family with sequence similarity 49 member B）是pol-miR-novel_171的靶基因，并受后者负调控。mRNA表达分析表明，牙鲆FAM49B（PoFAM49B）在多个组织中表达。重组的PoFAM49B蛋白（rPoFAM49B）能够与革兰氏阴性菌相互作用并抑制E. tarda和荧光假单胞菌的生长。在牙鲆细胞中干扰PoFAM49B表达能促进E. tarda在胞内的复制，过量表达pol-miR-novel_171对E. tarda的胞内复制也有类似的影响。同样，在牙鲆体内干扰PoFAM49B表达增强E. tarda在鱼体组织中的传播。此外，在牙鲆细胞中干扰PoFAM49B表达或过量表达pol-miR-novel_171促进细胞凋亡，而在体内和体外干扰PoFAM49B表达可提高促凋亡基因的表达。

        3、对于pol-miR-novel_547，我们发现其靶基因是PTEN（phosphatase and tensin homolog）。pol-miR-novel_547在mRNA和蛋白水平均抑制牙鲆PTEN（PoPTEN）的表达。E. tarda和RBIV-C1感染均调控pol-miR-novel_547和PoPTEN的表达。在牙鲆细胞中干扰PoPTEN表达或过量表达pol-miR-novel_547，都显著促进E. tarda的侵袭。同样，在牙鲆体内干扰PoPTEN表达能增强E. tarda的感染，而在体内过量表达PoPTEN则削弱了细菌的感染，但促进了RBIV-C1的复制。进一步的研究表明，PoPTEN通过AKT/mTOR通路影响自噬激活，并调节细胞凋亡。

        4、对于pol-miR-novel_395，我们发现其靶向PUF60（poly(U)-binding-splicing factor 60 kDa）。pol-miR-novel_395和牙鲆PUF60（PoPUF60）的表达均受E. tarda和RBIV-C1感染调控。E. tarda感染上调PoPUF60表达，而RBIV-C1感染下调PoPUF60表达。干扰PoPUF60或过表达pol-miR-novel_395显著增强E. tarda在牙鲆细胞中的复制。同样，在牙鲆体内干扰PoPUF60可以促进细菌在牙鲆组织中的传播，但阻滞RBIV-C1的复制；然而，在牙鲆体内过表达PoPUF60显著抑制细菌的传播，但促进病毒复制。

    综上所述，我们首次鉴定了E. tarda诱导的牙鲆肾脏miRNAs的表达图谱，揭示了pol-miR-182-5p、pol-miR-novel_171、pol-miR-novel_547和pol-miR-novel_395的调控机制，以及在病原感染、细胞自噬和细胞凋亡中的作用。这些结果为miRNAs介导的牙鲆抗菌免疫研究提供了新的见解。

    Japanese flounder (Paralichthys olivaceus) is an important aquaculture fish species farmed in China. It is susceptible to infection by several fish pathogen, including Edwardsiella tarda. E. tarda is a Gram-negative bacterium with a wide host range. MicroRNAs (miRNAs) are a type of single-stranded, non-coding RNAs consisting of 21-23 nucleotides and participate in the regulation of multi-biological processes, including pathogen infection, cellular autophagy, and cellular apoptosis, by negatively regulating the expression of their target genes at the post-transcriptional level. In this dissertation study, we employed high-throughput sequencing technology to examine miRNAs expression in the kidney of Japanese flounder infected with E. tarda at different time points. A total of 96 miRNAs showed differential expression (named DEmiRNAs) after E. tarda infection. The target gene prediction analysis was carried out for these 96 DEmiRNAs, and a total of 2779 potential target genes were found. Enrichment analysis of target genes revealed that these 2779 potential target genes were associated with diverse biological processes, cellular components, and molecular functions, and regulated diverse pathways related to immunity.

    Among the above 96 DEmiRNAs, four DEmiRNAs with large fold change or abundant expression, i.e. pol-miR-182-5p, pol-miR-novel_171, pol-miR-novel_547 and pol-miR-novel_395, were selected for the study of regulatory mechanisms during the infection of E. tarda.

    1. For pol-miR-182-5p, we found that the target gene of pol-miR-182-5p was sphingosine-1-phosphate receptor 1 (S1PR1). pol-miR-182-5p regulated Japanese flounder S1PR1 (PoS1PR1) negatively via the specific interaction with the 3′UTR of PoS1PR1. Overexpression of pol-miR-182-5p in flounder cells promoted apoptosis and inhibited cellular proliferation. Knockdown of PoS1PR1 in flounder enhanced E. tarda invasion and dissemination in fish tissues.

    2. For pol-miR-novel_171, we found that pol-miR-novel_171 expression was regulated by E. tarda and megalocytivirus (RBIV-C1) infection. Family with sequence similarity 49 member B (FAM49B) was the target gene of pol-miR-novel_171, and was negatively regulated by the latter. Japanese flounder FAM49B (PoFAM49B) expressed in multiple tissues of flounder, and recombinant PoFAM49B (rPoFAM49B) interacted with Gram-negative bacteria and inhibited the growth of E. tarda and Pseudomonas fluorescens. Interference with PoFAM49B expression in flounder cells promoted the intracellular replication of E. tarda. Similar effects on the intracellular replication of E. tarda were observed with pol-miR-novel_171 overexpression. Consistently, in vivo knockdown of PoFAM49B in flounder enhanced E. tarda dissemination in fish tissues. Furthermore, interference with PoFAM49B expression, or overexpression of pol-miR-novel_171 in flounder cells, promoted cellular apoptosis, while in vitro and in vivo knockdown of PoFAM49B augmented the expressions of pro-apoptotic genes.

    3. For pol-miR-novel_547, we found that the target gene of pol-miR-novel_547 is phosphatase and tensin homolog (PTEN). It negatively regulated the expression of Japanese flounder phosphatase and tensin homolog (PoPTEN) in mRNA and protein levels. E. tarda and RBIV-C1 could affect the expression of pol-miR-novel_547 and PoPTEN. Interference with PoPTEN expression or overexpression of pol-miR-novel_547 in flounder cells promoted E. tarda invasion. Consistently, in vivo knockdown of PoPTEN enhanced E. tarda dissemination in flounder tissues, whereas in vivo overexpression of PoPTEN attenuated E. tarda dissemination but facilitated RBIV-C1 replication. Further studies showed that PoPTEN affected autophagy activation via the AKT/mTOR pathway and also modulated the process of apoptosis.

    4. For pol-miR-novel_395, we found that it targeted the gene of poly (U)-binding-splicing factor 60 kDa (PUF60). The expression of pol-miR-novel_395 and Japanese flounder PUF60 (PoPUF60) was regulated by E. tarda and RBIV-C1, and constitutive expression of PoPUF60 expression occurred in relatively high levels in the heart and liver of flounder. E. tarda infection upregulated PoPUF60 expression, whereas RBIV-C1 infection downregulated PoPUF60 expression. Interference with PoPUF60 expression or overexpression of pol-miR-novel_395 in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoPUF60 enhanced bacterial dissemination in the tissues of flounder but blocked viral replication, whereas in vivo overexpression of PoPUF60 inhibited bacterial dissemination but facilitated viral replication.

    In summary, we obtained the first expression profile of E.tarda-induced host miRNAs expressed in Japanese flounder kidney, and revealed the regulatory mechanisms of pol-miR-182-5p, pol-miR-novel_171, pol-miR-novel_547 and pol-miR-novel_395 and their effects on pathogen infection, cellular autophagy, and cellular apoptosis. These results provided new insights into the mechanism of miRNA-mediated flounder immune defense against bacterial infection.

第1章 引言... 1

1.1 MicroRNA概述... 1

1.1.1 MicroRNA的发现... 1

1.1.2 MicroRNA的合成机制... 2

1.1.3 MicroRNA的调控机制... 3

1.1.4 MicroRNA的生物学功能... 5

1.1.5 MicroRNA在水产病害中的研究进展... 7

1.2 迟缓爱德华氏菌概述... 8

1.2.1 迟缓爱德华氏菌的感染途径... 9

1.2.2 迟缓爱德华氏菌的毒力机制和适应机制... 10

1.2.3 迟缓爱德华氏菌的免疫逃逸机制... 11

1.3 S1PR1（sphingosine-1-phosphate receptor 1）概述... 12

1.3.1 S1PR1对细胞迁移的调控... 12

1.3.2 S1PR1对细胞增殖和凋亡的调控... 13

1.3.3 S1PR1对病原感染的调控... 14

1.4 FAM49B（family with sequence similarity 49 member B）概述... 14

1.4.1 FAM49B对细胞迁移的调控... 15

1.4.2 FAM49B对细胞增殖、凋亡的调控... 16

1.4.3 FAM49B对免疫过程的调控... 16

1.5 PTEN（phosphatase and tensin homolog）概述... 16

1.5.1 PI3K/AKT信号通路... 17

1.5.2 PTEN对细胞周期和凋亡的调控作用... 18

1.5.3 PTEN对细胞自噬的调控作用... 19

1.6 PUF60（poly (U)-binding-splicing factor 60 kDa）概述... 20

1.6.1 PUF60对细胞凋亡的影响... 20

1.6.2 PUF60对免疫调控的影响... 20

1.7 本研究的目的与意义... 21

第2章 迟缓爱德华氏菌感染条件下牙鲆肾脏miRNA表达谱的鉴定... 22

2.1 材料和方法... 23

2.1.1 实验材料... 23

2.1.2 细菌感染和样品采集... 23

2.1.3 MiRNA高通量测序... 23

2.1.4 MiRNA测序结果分析... 23

2.1.5 MiRNA的靶基因预测和分析... 24

2.1.6 MiRNA的茎环反转录荧光定量PCR（stem-loop-qRT-PCR）验证... 24

2.2 结果... 26

2.2.1 迟缓爱德华氏菌感染后牙鲆miRNA的鉴定... 26

2.2.2 迟缓爱德华氏菌感染后牙鲆差异表达miRNA的鉴定... 28

2.2.3 牙鲆差异表达miRNA的靶基因预测... 35

2.2.4 牙鲆差异表达miRNA的靶基因的富集性分析... 42

2.2.5 牙鲆差异表达miRNA的qRT-PCR 验证... 44

2.3 讨论... 46

第3章 pol-miR-182-5p及其靶基因S1PR1的功能研究... 48

3.1 材料和方法... 48

3.1.1 实验材料... 48

3.1.2 体内感染实验... 48

3.1.3 MiRNA模拟物及转染... 49

3.1.4 pol-miR-182-5p对细胞凋亡影响的检测... 49

3.1.5 pol-miR-182-5p对细胞增殖影响的检测... 50

3.1.6 质粒构建... 50

3.1.7 荧光素酶报告实验... 58

3.1.8 PoS1PR1的序列分析... 58

3.1.9 牙鲆体内干扰PoS1PR1质粒效率的检测... 58

3.1.10在牙鲆体内干扰PoS1PR1后细菌感染实验... 59

3.1.11 统计分析... 59

3.2 结果... 59

3.2.1 迟缓爱德华氏菌/细胞肿大病毒对pol-miR-182-5p表达的影响... 59

3.2.2 pol-miR-182-5p对细胞凋亡和增殖的影响... 60

3.2.3 pol-miR-182-5p对PoS1PR1的调控作用... 62

3.2.4 PoS1PR1的序列比对及分析... 64

3.2.5 在牙鲆体内干扰PoS1PR1对迟缓爱德华氏菌在鱼组织内感染情况的影响... 66

3.3 讨论... 69

第4章 pol-miR-novel_171及其靶基因FAM49B的功能研究... 71

4.1 材料和方法... 71

4.1.1 实验材料... 71

4.1.2 体内感染实验... 72

4.1.3 MiRNA模拟物及转染... 72

4.1.4 体外干扰RNA及转染... 72

4.1.5 质粒构建... 73

4.1.6 荧光素酶报告实验... 75

4.1.7 PoFAM9B的序列分析... 75

4.1.8 PoFAM49B的组织特异性表达... 75

4.1.9 重组PoFAM9B（rPoFAM9B）的表达、纯化及透析... 75

4.1.10 rPoFAM9B与细菌的相互作用的检测... 77

4.1.11 rPoFAM9B对细菌生长的影响的检测... 78

4.1.12 pol-miR-novel_171和PoFAM49B对迟缓爱德华氏菌胞内复制能力的检测... 78

4.1.13 牙鲆体内干扰PoFAM9B质粒效率的检测... 79

4.1.14 在牙鲆体内干扰PoFAM49B后细菌感染实验... 79

4.1.15 pol-miR-novel_171和PoFAM49B对细胞凋亡率影响的检测... 79

4.1.16 干扰PoFAM9B后，促凋亡基因表达水平的检测... 79

4.2 结果... 79

4.2.1 迟缓爱德华氏菌/细胞肿大病毒对pol-miR-novel_171表达的影响... 79

4.2.2 pol-miR-novel_171对PoFAM49B的调控作用... 81

4.2.3 PoFAM49B的序列分析和组织特异性表达... 83

4.2.4 rPoFAM49B与细菌的相互作用及其对细菌生长的影响... 85

4.2.5 pol-miR-novel_171和PoFAM49B对迟缓爱德华氏菌胞内复制的影响... 88

4.2.6干扰PoFAM49B对迟缓爱德华氏菌感染牙鲆的影响... 89

4.2.7 pol-miR-novel_171和PoFAM49B对细胞凋亡的影响... 91

4.3 讨论... 94

第5章 pol-miR-novel_547及其靶基因PTEN的功能研究... 97

5.1 材料和方法... 97

5.1.1 实验材料... 97

5.1.2 体内感染实验... 97

5.1.3 MiRNA模拟物、miRNA抑制剂及转染... 97

5.1.4 体外干扰RNA及转染... 97

5.1.5 质粒构建... 98

5.1.6 荧光素酶报告实验... 100

5.1.7 Western blot 实验... 100

5.1.8 PoPTEN的序列分析... 101

5.1.9 PoPTEN的组织特异性表达... 101

5.1.10 pol-miR-novel_547和PoPTEN对迟缓爱德华氏菌胞内复制能力的检测... 101

5.1.11 牙鲆体内干扰PoPTEN质粒效率的检测... 101

5.1.12 在牙鲆体内干扰PoPTEN后细菌感染实验... 101

5.1.13 牙鲆体内过表达PoPTEN质粒效率的检测... 101

5.1.14 在牙鲆体内过表达PoPTEN后细菌感染实验... 102

5.1.15 在牙鲆体内过表达PoPTEN后病毒感染实验... 102

5.1.16 在牙鲆细胞中过表达pol-miR-novel_547或干扰PoPTEN后，自噬相关基因表达水平的检测    103

5.1.17 在牙鲆体内干扰或过表达PoPTEN后，自噬相关基因表达水平的检测... 103

5.1.18 pol-miR-novel_547和PoPTEN对哺乳动物和鱼类细胞自噬影响的检测... 103

5.1.19 pol-miR-novel_547和PoPTEN对细胞凋亡影响的检测... 103

5.2 结果... 104

5.2.1 迟缓爱德华氏菌/细胞肿大病毒对pol-miR-novel_547表达的影响... 104

5.2.2 pol-miR-novel_547对PoPTEN的调控作用... 105

5.2.3 PoPTEN的序列分析和组织特异性表达... 107

5.2.4 迟缓爱德华氏菌/细胞肿大病毒对PoPTEN表达的影响... 109

5.2.5 PoPTEN对病原感染的影响... 109

5.2.6 PoPTEN对自噬的影响... 114

5.2.7 pol-miR-novel_547和PoPTEN对细胞凋亡的影响... 117

5.3 讨论... 118

第6章 pol-miR-novel_395及其靶基因PUF60的功能研究... 122

6.1 材料和方法... 122

6.1.1 实验材料... 122

6.1.2 体内感染实验... 122

6.1.3 MiRNA模拟物及转染... 122

6.1.4 体外干扰RNA及转染... 122

6.1.5 质粒构建... 123

6.1.6 荧光素酶报告实验... 124

6.1.7 PoPUF60的序列分析... 125

6.1.8 PoPUF60的组织特异性表达... 125

6.1.9 pol-miR-novel_395和PoPUF60对迟缓爱德华氏菌胞内复制能力的检测... 125

6.1.10 牙鲆体内干扰PoPUF60质粒效率的检测... 125

6.1.11 在牙鲆体内干扰PoPUF60后细菌感染实验... 125

6.1.12 在牙鲆体内干扰PoPUF60后病毒感染实验... 125

6.1.13 牙鲆体内过表达PoPUF60质粒效率的检测... 125

6.1.14 在牙鲆体内过表达PoPUF60后细菌感染实验... 126

6.1.15 在牙鲆体内过表达PoPUF60后病毒感染实验... 126

6.2 结果... 126

6.2.1 pol-miR-novel_395对PoPUF60的调控作用... 126

6.2.2 PoPUF60的序列分析和组织特异性表达... 128

6.2.3 迟缓爱德华氏菌/细胞肿大病毒对PoPUF60及pol-miR-novel_395表达的影响... 130

6.2.4 PoPUF60和pol-miR-novel_395对病原感染的影响... 131

6.3 讨论... 136

第7章 结论与展望... 138

参考文献... 140

附录一 实验所用溶液的配制方法... 174

附录二 实验所用引物序列... 178

附录三 实验所用仪器设备... 184

致 谢... 185

作者简历及攻读学位期间发表的学术论文     186