实验海洋生物学重点实验室知识库

  文蛤是一种重要的海洋经济贝类，近年来由于养殖环境的恶化和病原菌的侵袭，文蛤的爆发性死亡频频发生，已成为制约文蛤养殖业发展的一个重要问题，因此解析文蛤感染副溶血弧菌后的抗病机制、培育抗病品种，是解决这一问题的一个有效途径。随着对抗病性状研究的深入，人们发现文蛤感染副溶血弧菌后能否生存不仅与宿主免疫系统防御有关，还与其能量、物质代谢调控紧密相关。因此研究感染后宿主体内能量代谢调控，对于深入理解文蛤免疫防御之下的能量、物质代谢变化机制与存活或死亡之间的关系具有重要意义。本研究围绕文蛤能量代谢调控中心MpAMPK以及关键能量代谢途径-脂质代谢，探讨副溶血弧菌感染过程中文蛤能量代谢调控对副溶血弧菌感染的响应，揭示了能量代谢在宿主应对副溶血弧菌感染调控中的关键作用，在此基础上利用抗性和敏感性家系材料，筛选到了能量代谢相关的弧菌抗性候选基因，其基础表达水平与感染后存活率密切相关，以上研究为抗病品种选育提供了新的分子标记。主要结果如下：

1、获得了文蛤MpAMPK-α2序列，经过同源序列分析，MpAMPK-α2序列具有保守性。通过副溶血弧菌人工感染实验，明确了肌肉组织中的AMP/ATP、ADP/AMP对副溶血弧菌感染的响应，与之相对应，检测到MpAMPK在基因表达水平和磷酸化水平对AMP/ATP、ADP/AMP指标变化的应答。进一步探究了副溶血弧菌感染过程中MpAMPK 下游的糖、脂肪和蛋白质代谢调控以及NAD⁺/NADH的变化趋势，明确了糖酵解过程加强，并且糖、蛋白和脂肪代谢呈现出合成代谢增强的趋势，与之相应NAD⁺/NADH比值呈现上调趋势。以上结果表明文蛤应对副溶血弧菌感染是一个耗能的过程，宿主可能通过增强糖酵解和糖、蛋白和脂质的合成来满足免疫防御及损伤修复过程中的能量和物质供给。

2、根据转录组数据提示，副溶血弧菌感染对文蛤体内的能量代谢相关途径基因的表达均产生显著影响。因此本部分重点关注了感染过程中文蛤肝胰腺脂质代谢途径中甘油三酯合成和分解代谢、脂肪酸从头合成代谢以及脂肪酸β氧化过程中关键酶基因表达及关键产物含量指标的变化，结果表明，副溶血弧菌感染后文蛤肝胰腺的甘油三酯（TG）含量降低，游离脂肪酸（FFA）含量升高。相应地，TG的合成关键酶活性显著降低，FFA从头合成增强相关关键酶表达增强，但脂肪酸β氧化途径的关键基因没有明显变化。以上结果支持了脂质代谢变化与副溶血弧菌感染后文蛤死亡的关联性，有助于更好地理解脂类代谢在宿主应对病原刺激时能量调控的作用。

3、利用副溶血弧菌人工感染，根据死亡率的差异鉴定出抗性群体和敏感群体。在明确糖、脂肪和蛋白代谢对副溶血弧菌感染存在明显响应的基础之上，探究副溶血弧菌感染前后这三个主要能量代谢途径的14个关键基因在抗性和敏感性群体中的表达差异。结果发现10个基因（GS、mTOR、eEF2K、ATGL、MDY、SREBP、FAS、CPT-1、ACS和ECH）在未感染状态下的基础表达水平和9个基因（GS、mTOR、eEF2K、SREBP、FAS、ACC、CPT-1、ACAD和ECH）在感染条件下的诱导表达水平在不同抗性群体中具有显著差异，且抗性群体均具有更高的表达水平，提示较高的代谢水平可能是抗性群体的代谢特点。其中10个基因（GS、mTOR、eEF2K、ATGL、MDY、SREBP、FAS、CPT-1、ACS和ECH）在未感染状态下的基础表达水平可以作为判断文蛤副溶血弧菌抗性能力的潜在标记，为文蛤抗性选育提供理论和技术支持。

  Meretrix petechialis is an important marine economic shellfish. In recent years, due to the deterioration of the breeding environment and the invasion of pathogenic bacteria, massive death of M.petechialis occurs frequently, which has become an important problem restricting the development of M.petechialis aquaculture industry. Therefore, analysing the disease resistance mechanism of M.petechialis infected Vibrio parahaemolyticus and selecting the Vibrio-resistant varieties is an effective way to solve this problem. With the further study of Vibrio resistance traits, it has been found that the survival of M. petechialis infected V.parahaemolyticus is not only related to the defense of host immune system, but also closely related to the regulation of energy and material metabolism during V.parahaemolyticus infection. Therefore, it is significant to understand the relationship between the mechanism of energy and material metabolism changes and fate of survival or death during V.parahaemolyticus infection. This study focuses on the energy metabolism regulation center MpAMPK of M.petechialis and the key energy metabolism pathway-lipid metabolism, and investigates the energy metabolism response of M.petechialis to V.parahaemolyticus infection revealing the key role of energy metabolism in response to V.parahaemolyticus infection. Then, candidate genes related to energy metabolism were screened for resistant biomarker, based on resistant and sensitive clam families, and the basic expression level of these genes is closely related to the survival rate after infection. The above research provides new molecular markers for the Vibrio-resistant clam selection. The main results are as follows:

1. The MpAMPK-α2 sequence of M.petechialis was obtained. It is found that the sequence of MpAMPK-α2 has a degree of conservation by homologous sequence analysis. The response of AMP/ATP and ADP/AMP in muscle tissue of M.petechialis were clarified to V.parahaemolyticus infection by artificial V.parahaemolyticus infection experiment. Correspondingly, it was detected that MpAMPK gene expression and P-MpAMPK activity had a positive response on AMP/ATP and ADP/ATP level. We further explored metabolic regulation of glucose, lipid and protein downstream of MpAMPK and the change of NAD⁺/NADH value in the process of V.parahaemolyticus infection. It is clear that glycolysis process enhanced and the metabolism of glucose, protein and lipid showed a trend of enhanced anabolism, which was also aligned to the increase of NAD⁺/NADH ratio. These results suggested that coping with V.parahaemolyticus infection for M.petechialis is an energy-consuming process, and the host may enhance glycolysis process, and glucose, protein and lipid synthesis to meet the energy and material supply in the establishment of immune defense and damage repair during V.parahaemolyticus infection.

2. It was suggested that V.parahaemolyticus infection has a significant effect on energy metabolism-related gene expression in M.petechialis based on transcriptome data. So this part of the study focused on the changes of key enzyme gene expression and key content indicators of products in triglyceride anabolism and catabolism, fatty acid de novo synthesis, and fatty acid β oxidation of lipid metabolism pathway in the hepatopancreas of M. petechialis during V.parahaemolyticus infection. The results show that levels of triglycerides (TG) decreased and free fatty acids (FFA) increased in the infected clams. Correspondingly, the synthase activities of TG were significantly reduced after V.parahaemolyticus infection. Additionally, expression of key enzymes related to FFA de novo synthesis was strengthened, but key genes of the fatty acid β-oxidation pathway showed no significant changes after V.parahaemolyticus infection. The results supported the relationship existed between lipid metabolism changes and the emergence of massive mortality induced by bacterial infection in clams, which will help to better understand the role of lipid metabolism in energy regulation when the host responds to V.parahaemolyticus infection.

3. The resistant and sensitive clam populations were identified based on the mortality difference by V.parahaemolyticus infection experiment. Based on the significant response of glucose, lipid and protein metabolism to V.parahaemolyticus infection, we examined the mRNA expression of the 14 key genes of these three main energy metabolism pathways in the resistant and susceptible groups kept in both uninfected and infected conditions. The basic expression levels of 10 genes (GS, mTOR, eEF2K, ATGL, MDY, SREBP, FAS, CPT-1, ACS and ECH) in the uninfected state and 9 genes (GS, mTOR, eEF2K, SREBP, FAS, ACC, CPT-1, ACAD and ECH) under infection condition were significantly different, and the expression in the resistant group was higher. It revealed that the higher metabolic level may be the metabolic characteristic of the resistant group. Among them, the gene expression of 10 genes (GS, mTOR, eEF2K, ATGL, MDY, SREBP, FAS, CPT-1, ACS and ECH) in the uninfected state can be used to determine different Vibrio resistance of M. petechialis without V.parahaemolyticus challenge as potential markers, which provide theoretical and technical support for the breeding of Vibrio-resistant clams.

第1章 引言………………………………………………………………………..1

1.1 文蛤生物学及养殖现状 ……………………………………………………1

1.2 贝类的免疫防御特征及其与死亡的关系 …………………………………2

1.3 免疫代谢与健康的关系…………………………………………………….3

1.4 贝类的能量代谢调控过程………………………………………………….5

1.4.1 贝类能量代谢关键因子AMPK………………………………………….5

1.4.2 脂类代谢调控……………………………………………………………..6

1.4.3 糖代谢和蛋白质代谢调控 ……………………………………………….7

1.5 本研究的内容和意义……………………………………………………….9

第2章 文蛤MpAMPK为中心的能量代谢对副溶血弧菌感染的响应….............................................................................................................................11

2.1 研究背景…………………………………………………………………...11

2.2 材料与方法………………………………………………………………...12

2.2.1 副溶血弧菌感染与组织取样……………………………………………12

2.2.2 RNA提取和cDNA合成…………………………………………………13

2.2.3 文蛤MpAMPK-α2基因cDNA克隆与序列分析………………………14

2.2.4 qRT-PCR…………………………………………………………………..15

2.2.5 Western blot 分析………………………………………………………...16

2.2.6 HPLC-MS/MS检测………………………………………………………17

2.2.7 磷酸果糖激酶（PFK）活性检测……………………………………….18

2.2.8 糖原含量和游离脂肪酸（FFA）含量检测…………………………….18

2.3 结果………………………………………………………………………...19

2.3.1 MpAMPK-α2基因的序列分析…………………………………………..19

2.3.2 MpAMPK-α2的组织表达和对副溶血弧菌感染的响应………………..20

2.3.3 副溶血弧菌感染过程中文蛤肌肉组织能量水平变化和MpAMPK磷酸化响应………………………………………………………………………………..22

2.3.4 副溶血弧菌感染后文蛤肌肉组织中糖代谢的变化……………………24

2.3.5 副溶血弧菌感染过程中文蛤肌肉组织中蛋白、脂肪合成过程以及NAD+/NADH的变化………………………………………………………………..26

2.4 讨论………………………………………………………………………...29

第3章 文蛤肝胰腺中脂类代谢对副溶血弧菌感染过程的响应特征…………………………………………………………………………………….33

3.1 研究背景…………………………………………………………………..33

3.2 材料与方法………………………………………………………………..34

3.2.1 第一次副溶血弧菌感染实验和组织取样用于转录组分析和基因表达定量……………………………………………………………………………………..34

3.2.2 第二次副溶血弧菌感染实验和组织取样用于生化分析……………....36

3.2.3 转录组分析和数据再挖掘………………………………………………36

3.2.4 RNA提取和基因表达分析………………………………………………36

3.2.5 文蛤肝胰腺中甘油三酯（TG）和游离脂肪酸（FFA）含量检测…………37

3.2.6 肝胰腺中肉毒碱棕榈酰转移酶（CPT-1）的活性检测…………………..38

3.3 结果………………………………………………………………………...38

3.3.1 副溶血弧菌感染后文蛤转录组中的脂质代谢数据分析………………38

3.3.2 甘油三酯合成和分解代谢对副溶血弧菌感染的响应…………………40

3.3.3 脂肪酸从头合成对副溶血弧菌感染的响应 …………………………...42

3.3.4 脂肪酸β氧化对副溶血弧菌感染的响应………………………………43

3.4 讨论………………………………………………………………………...45

第4章 代谢相关基因在抗性和敏感群体的差异表达及抗性标记筛选…………………………………………………………………………………….49

4.1 研究背景…………………………………………………………………...49

4.2 材料方法…………………………………………………………………...50

4.2.1 副溶血弧菌感染实验和组织取样………………………………………50

4.2.2 RNA提取和基因表达分析 ……………………………………………...51

4.3 结果………………………………………………………………………...51

4.3.1 糖代谢相关基因在不同抗性文蛤中的表达差异………………………51

4.3.2 蛋白代谢相关基因在不同抗性文蛤中的表达差异……………………52

4.3.3 脂质代谢相关基因在不同抗性文蛤中的表达差异……………………53

4.4 讨论………………………………………………………………………...57

结论………………………………………………………………….…………..…..61

参考文献……………………………………………………………………….…..63

附录………………………………………………………………………............…81

致谢……………………………………………………………………….………...85

作者简历及攻读学位期间发表的学术论文与研究成果…………………………………………………………………………………….87