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文蛤对副溶血性弧菌感染的免疫和代谢响应及关键调控通路解析
于佳佳
Subtype博士
Thesis Advisor刘保忠
2020-05-13
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name农学博士
Keyword文蛤 转录组 弧菌载量 爆发性死亡 免疫和代谢响应
Abstract

文蛤(Meretrix petechialis)是一种重要的经济水产养殖贝类。在文蛤养殖过程中,弧菌感染引起的爆发性死亡事件时有发生。因此,了解疾病发生和致病机理,可以为病害防治和抗性品种选育提供科学基础。本研究利用副溶血性弧菌(Vibrio parahaemolyticus)对文蛤进行人工模拟攻毒感染实验,通过转录组分析了弧菌感染过程中文蛤肝胰腺免疫和代谢响应,研究了弧菌感染早期肝胰腺载菌量和免疫通路变化,对死亡爆发期相关的糖异生过程关键通路进行了鉴定,分析了弧菌攻毒强度与肝胰腺弧菌载量相关性。主要的研究结果如下:

1、对文蛤进行副溶血性弧菌浸泡感染实验,基于文蛤死亡率数据将感染过程分为潜伏期、前驱期、爆发期和恢复期等四个阶段。利用转录组研究了文蛤肝胰腺在不同感染阶段对感染的动态响应,在四个感染阶段共得到了38067个差异表达基因(DEGs)。DEGs注释表明免疫相关和代谢相关信号通路显著富集,免疫防御和代谢调控在病原感染过程中发挥关键作用。基于时间序列的聚类分析,DEGs基因的表达类型可分为三类,即U形、L形和倒V形;对三种表达类型基因进行KEGG分析,显示感染期间合成代谢和细胞生长增殖相关信号通路被长期或短暂抑制,免疫相关信号通路呈现针对细菌感染的诱导表达或抑制表达,表明免疫系统采取不同的策略来抵御细菌感染。此外,一些信号通路如PI3K-Akt通路可同时参与免疫防御和细胞代谢,表明免疫系统和代谢系统能相互协调响应细菌感染。

2、实验分析了弧菌感染早期(即前期和潜伏期)文蛤肝胰腺中弧菌载量的变化趋势,表明弧菌载量在第1天维持较高水平,第2天显著下降,随后维持较低水平。转录组分析结合定量PCR结果表明Toll通路、Imd通路和ROS系统在早期感染过程中发挥了重要的免疫防御作用,宿主通过激活上调Toll通路和ROS系统分别释放抗菌肽和过氧化氢,并可能通过抑制Imd通路抑制病原菌的增殖。RNAi结果进一步支持Toll通路和ROS系统分别通过调控抗菌肽和过氧化氢的生成参与杀菌抑菌过程。此外,筛选验证了上述通路/系统中表达量与肝胰腺弧菌载量相关的基因,为文蛤抗弧菌品系的选育提供了候选分子标记。

3、通过对弧菌感染过程中死亡爆发期文蛤肝胰腺的转录组数据分析,发现PPARAMPKPI3K-Akt信号通路以及糖酵解/糖异生等代谢相关信号通路被显著富集,表明代谢相关通路参与了宿主免疫过程。加权基因共表达网络分析(weighted correlation network analysisWGCNA)显示,死亡爆发期富集到糖异生过程的关键酶基因,表明糖异生过程与文蛤爆发性死亡事件相关;通过检测糖异生过程中代谢标志物葡萄糖和磷酸烯醇丙酮酸羧激酶(phosphoenolpyruvate carboxykinasePEPCK)等含量变化以及糖异生过程相关基因葡萄糖-6-磷酸酶(glucose-6-phosphataseG6PasePEPCK等的表达变化,发现糖异生在感染过程中被抑制。进一步分析文蛤濒死和存活个体中代谢标志物含量和PI3K-Akt信号通路中关键基因的表达差异,支持了糖异生过程与爆发性死亡事件的关联性。该结果有助于我们更好地理解文蛤爆发性死亡过程的分子机制,并为贝类健康状况的检测提供基因标记。

4、用四个不同浓度的副溶血性弧菌,对两份具有不同存活率的文蛤家系group Agroup B进行攻毒感染实验,发现在四个弧菌浓度下,group A的存活率均显著高于group B;文蛤存活率随着弧菌攻毒浓度的增加而降低,但出现死亡和达到死亡高峰的时间大体一致。不同处理浓度下,肝胰腺中弧菌载量在时间序列上呈现相同的变化趋势,在3 dpi文蛤肝胰腺弧菌载量随着弧菌感染浓度的升高而升高。基于肝胰腺中弧菌载量对两组文蛤的抗性(清除弧菌能力)进行评估,group A为高抗而group B为低抗家系,与攻毒存活率结果一致;检测了(dual oxidaseDuox髓样分化蛋白抗原(myeloid differential protein-88Myd88Tube和过氧化氢酶(catalaseCAT)等免疫相关基因在不同存活率文蛤个体中的表达及其与弧菌载量变化的相关性,筛选了可作为文蛤弧菌抗性的指示标记。

Other Abstract

The clam Meretrix petechialis is an important commercial aquaculture species in China. During the clam culture period, mass mortality events often occur due to the Vibrio infection. Understanding the mechanism of disease occurrence and host immune response can provide a scientific basis for disease control and genetic breeding of resistance varieties. In this study, M. petechialis was challenged with Vibrio parahaemolyticus immersion to simulate a natural infection, and the dynamic immune and metabolism response of clam hepatopancreas to Vibrio infection was analyzed by transcriptome analysis. We investigated the variation of hepatopancreas Vibrio load and immune-related signaling pathways during early bacterial infection, and identified the key signaling pathways related to gluconeogenesis at death outbreak phase. In addition, we analyzed the correlation between the infection doses and the Vibrio load. The main results were as follows:

After M. petechialis were challenged with V. parahaemolyticus immersion, the infection process was divided into four phases including latency, prodrome, onset and recovery phases based on the clam mortality data. The dynamic response of clams to Vibrio infection at different infection phases were investigated by transcriptome analysis. A total of 38067 differentially expressed genes (DEGs) were identified at different infection phases. DEG annotations showed that immune-related and metabolism-related signaling pathways were enriched, indicating that immune defense and metabolism process play key roles during bacterial infection. Three kinds of expression pattern were classified by cluster analysis, including U-shape, L-shape and inverted V-shape, and the KEGG analysis were performed, respectively. Anabolism and cellular growth proliferation related signaling pathways were repressed (long-lasting or transient) during bacterial infection. However, the immune related signaling pathways with different immune functions showed induction expression or repression expression against bacterial infection, which indicated that immune system take different strategies against bacterial infection. Furthermore, some signaling pathways such as PI3K-Akt signaling pathway both involved in immune defense and cell metabolism. This study provides a sight that the dynamic immunity and metabolic responses may be integrated to improve the host survival and shift more energy for immune defense.

In this study, the Vibrio load variation in hepatopancreas were analyzed during early phase of Vibrio infection (i.e., latency, prodrome) and the results showed that Vibrio load was high at 1 dpi (days post-infection), and then dramatically decreased at 2 dpi and kept low level at following time points. The results of transcriptome analysis and quantitative real time PCR indicated the Toll and Imd pathway and ROS (reactive oxygen) system play important immune defense roles during early bacterial infection. The Toll pathway and ROS system were activated to release antibacterial peptide (AMP) and H2O2, respectively, while the Imd pathway might be repressed to prevent from the bacteria proliferation. The RNAi experiment further supported that Toll pathway and ROS system could directly control the Vibrio abundance via producing AMP and H2O2. In addition, we identified the Vibrio-load related genes in the above pathways, which provide the candidate molecular markers for genetic breeding of Vibrio-resistant clam strain.

The transcriptome analysis of clam hepatopancreas during the death outbreak phase showed the significantly enriched of multiple metabolism-related signaling pathways containing PPAR, AMPK, PI3K-Akt signaling pathways and glycolysis/ gluconeogenesis, which revealed the involvement of metabolism-related signaling pathways in host immune defense. The weighted correlation network analysis (WGCNA) exhibited that several key enzymes involved in gluconeogenesis were enriched at this phase, indicating that the gluconeogenesis was related to death outbreak of clams. The detection of content change of metabolic markers (i.e., glucose and phosphoenolpyruvate carboxykinase (PEPCK)) and expression variation of key genes (i.e., glucose-6-phosphatase (G6Pase), PEPCK and etc.) in gluconeogenesis showed that the gluconeogenesis was repressed during Vibrio infection. Further analysis of the differences of metabolic markers and key genes in PI3K-Akt signaling pathway between moribund and survival individuals implied that gluconeogenesis was related with clam mass mortality. The results could help us to understand the molecular mechanism of death outbreak event and provide gene marker for healthy status detection of this clam.

Two clam groups with different survival rates (group A and group B) were challenged with four Vibrio doses and the result confirmed that group A had higher survival rates than group B in all the four doses. The survival rate decreased with the increase of the infection dose while the begin time and peak of death were similar under different doses. The change tendency of Vibrio load in hepatopancreas were similar during infection period among different infection doses, and Vibrio load increased with the infection dose increment at 3 dpi. The clam resistance (bactericidal ability) was evaluated based on the Vibrio load, group A is high resistant family and group B is low resistant family, respectively, which is consistent with the survival rate. In addition, we detected the expression level of immune-related genes containing dual oxidase (Duox), myeloid differential protein-88 (Myd88), Tube and catalase (CAT) among the individuals in group A and group B and its’ correlation with Vibrio load variation and screened several molecular indicators for clam resistance.

MOST Discipline Catalogue农学
Language中文
Table of Contents

目 录

 

 

第1章 引言... 1

 

1.1 文蛤生物学... 1

 

1.2 文蛤的经济价值及养殖现状... 1

 

1.3 贝类的免疫防御系统... 2

 

1.3.1 贝类防御系统组成... 3

 

1.3.2 贝类防御系统特点... 8

 

1.4 贝类代谢过程... 9

 

1.4.1 糖代谢... 10

 

1.4.2 脂类代谢... 11

 

1.5 免疫和代谢互作... 12

 

1.6 本研究的内容和意义... 12

 

第2章 文蛤在弧菌感染过程中肝胰腺免疫和代谢响应的组学分析... 14

 

2.1 研究背景... 14

 

2.2 材料与方法... 15

 

2.2.1 弧菌感染和样品收集... 15

 

2.2.2 文库构建和测序... 16

 

2.2.3 De novo转录组组装和基因功能注释... 16

 

2.2.4 测序比对和基因表达定量... 17

 

2.2.5 差异表达基因(DEGs)分析... 17

 

2.2.6 基因表达分析... 17

 

2.3 结果... 20

 

2.3.1 副溶血性弧菌感染后文蛤死亡率... 20

 

2.3.2 De novo转录组组装和基因注释... 21

 

2.3.3 响应弧菌感染的转录组数据分析... 22

 

2.3.4 基于时间序列的DEGs聚类分析... 26

 

2.3.5 不同表达模式中DEGs的作用... 27

 

2.3.6 DEGs可靠性验证... 30

 

2.4 讨论... 32

 

第3章 文蛤在弧菌感染早期肝胰腺载菌量变化和免疫关键通路解析... 35

 

3.1 研究背景... 35

 

3.2 材料与方法... 36

 

3.2.1 弧菌感染实验与样品收集... 36

 

3.2.2 涂板计数... 37

 

3.2.3 qRT-PCR.. 37

 

3.2.4 Western blot 分析... 40

 

3.2.5 细菌荧光原位杂交(FISH)... 41

 

3.2.6 组织切片荧光原位杂交... 42

 

3.2.7 RNA干扰... 42

 

3.3 结果... 44

 

3.3.1 弧菌感染早期文蛤肝胰腺内弧菌载量变化... 44

 

3.3.2 弧菌感染早期免疫相关信号通路的转录组分析... 48

 

3.3.3 弧菌感染早期Toll通路、Imd通路和ROS组分的变化... 50

 

3.3.4 Myd88,Dredd和Duox基因的功能分析... 53

 

3.3.5 文蛤肝胰腺弧菌载量相关基因的筛选验证... 56

 

3.4 讨论... 59

 

第4章 文蛤弧菌感染爆发性死亡相关的糖异生抑制和关键通路解析... 63

 

4.1 研究背景... 63

 

4.2 材料与方法... 64

 

4.2.1 转录组数据分析... 64

 

4.2.2 弧菌攻毒实验与样品收集... 64

 

4.2.3 RNA提取和cDNA合成... 65

 

4.2.4 qRT-PCR.. 65

 

4.2.4 葡萄糖含量及PEPCK活性检测... 66

 

4.3 结果... 66

 

4.3.1 转录组数据分析... 66

 

4.3.2 WGCNA分析... 69

 

4.3.3 感染死亡过程文蛤肝胰腺糖异生相关产物和酶的变化... 72

 

4.3.4 感染死亡过程糖异生相关基因的表达变化... 73

 

4.3.5 PI3K-Akt信号通路基因在不同健康状态文蛤中的表达差异... 74

 

4.4 讨论... 75

 

第5章 不同攻毒强度文蛤肝胰腺弧菌载量变化与免疫相关基因表达... 78

 

5.1 研究背景... 78

 

5.2 材料与方法... 79

 

5.2.1 实验动物... 79

 

5.2.2 弧菌感染实验与样品收集... 79

 

5.2.3 文蛤肝胰腺涂板计数... 80

 

5.2.4 RNA提取和cDNA合成... 80

 

5.2.5 qRT-PCR.. 80

 

5.3 结果... 80

 

5.3.1 不同文蛤材料攻毒后的存活率... 80

 

5.3.2 两组文蛤在不同攻毒浓度下的存活率比较... 81

 

5.3.3 两组文蛤在不同攻毒浓度下肝胰腺弧菌载量的时序变化... 83

 

5.3.4 两组文蛤弧菌抗性(清除弧菌能力)的量化评估... 85

 

5.3.5 免疫相关基因的表达差异及其与弧菌载量变化的相关分析... 88

 

5.4 讨论... 90

 

结 论... 93

 

参考文献... 95

 

附 录... 117

 

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

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164700
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
于佳佳. 文蛤对副溶血性弧菌感染的免疫和代谢响应及关键调控通路解析[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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