IOCAS-IR
生殖过程对文蛤免疫和代谢影响的研究
王迪
Subtype博士
Thesis Advisor刘保忠
2022-05-16
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Keyword文蛤 生殖 免疫 环境胁迫 TLR 通路
Abstract

文蛤(Meretrix petechialis)是我国重要的海产经济贝类。由于滩涂环境和气 候变化,在养殖过程中时有规模性死亡发生,给养殖产业带来严重的经济损失。 文蛤的规模性死亡多发生在夏季高温期,同时夏季也是文蛤的生殖季节。因为动 物的生殖和对病原的免疫响应都是高能量需求过程,因此文蛤生殖与免疫力之间 可能存在权衡。本研究针对贝类夏季死亡这一问题,选取生殖前后的文蛤亲本进 行了转录组和蛋白组学分析,解析生殖过程对免疫能力和物质能量代谢的影响。 另外,通过攻毒实验多角度探究了高温和弧菌病原对文蛤生存、免疫功能、氧化 应激以及对凋亡自噬的影响。基于组学数据,选取受生殖影响的 TLR 信号通路, 分析了其免疫功能和作用分子机制。本研究的主要结果如下:

1、为了研究繁殖过程对文蛤免疫的影响,我们选取生殖前后文蛤样品进行 了转录组和基因表达分析。基于 BGISEQ-500 测序平台共得到 48339 条 unigenes。 雌性生殖前后比较组(Pre_F-vs.-Post_F)共检测到差异基因(DEGs)1805 个, 而雄性生殖前后比较组(Pre_M-vs.-Post_M)共检测到 2488 个 DEGs。对 DEGs 进 行 KEGG 富集分析,显示雄雌共有的免疫信号通路在生殖后发生了改变,包括 IL-17 信号通路、TLR 信号通路、RIG-I 样受体信号通路和 NOD 样受体信号通路 等。通过 Nr 注释进一步筛选了 22 个免疫相关的 DEGs,它们分别来自补体与凝 血级联通路、细胞凋亡通路、TLR 信号通路、NF-κB 信号通路、白细胞跨内皮 迁移通路和 MAPK 信号通路等。进一步分析发现,雌性产卵后凋亡、TLR 信号 通路和热休克相关基因表达上调,而雄性生殖后补体相关基因表达下调。另外, 我们对产卵前后文蛤进行了攻毒实验,检测了四个基因(ARAF-like,CREM, TLR4 和 TBK1)在弧菌感染下生殖前/后的表达情况,发现雄雌蛤生殖后对弧菌 刺激的免疫应答水平均有上调。利用两个标记基因 DOUX 和 MITF 进一步评价 了文蛤生殖前、后样品在弧菌攻毒条件下的免疫状况,发现生殖后样品中 DOUX 和 MITF 在转录和蛋白水平上显著上调,进一步证实生殖影响了文蛤对病原菌刺 激的免疫响应水平。上述结果为探究生殖对文蛤免疫影响的分子机制提供了新认 知。

2、由于繁殖需要大量的能量投入,因此繁殖对代谢的影响日益受到关注。 本研究采用蛋白质组学方法,获得了雌性和雄性文蛤生殖前后的肝胰腺蛋白质组 数据。基于 iTRAQ 分析共得到 5,192 个蛋白,其中分别在雌性和雄性生殖前/后 样品检测到 42 个和 37 个差异蛋白(DEPs)。通过 KEGG 通路分析,探讨了获 得的 DEPs 的功能,结果表明生殖对代谢过程有重要影响。雌性糖原去分支酶 (AGL)、雄性甘露糖-6-磷酸异构酶(MPI)、β-葡萄糖苷酸酶(GUSB)和乙 醛酸酯/羟丙酮酸酯还原酶(GRHPR)在生殖后降低,意味着生殖后碳水化合物 代 谢 减 弱 。 雌 性 醛 脱 氢 酶 ( NAD+ ) 生 殖 后 上 调 , 雄 性 Meprin B 、 Cysteine-S-conjugateβ-lyase 裂合酶生殖后表达增加,提示生殖使得蛋白质和氨基 酸代谢增强。此外,谷胱甘肽转移酶/脱氢酶(DHAR)和谷胱甘肽 S-转移酶(GST) 在雌性生殖后个体上调,而在雄性生殖后个体下调,说明生殖导致雌性比雄性更 严重的氧化损伤。雌性的两种肌钙蛋白 troponin 和 myosin 以及 EFHD2 在生殖后 的表达下调,说明生殖可能会减弱雌性的肌肉功能。进一步实验显示,总葡萄糖 和蛋白质含量在生殖后短暂增加,然后恢复到基线水平。研究结果有助于更好地 理解文蛤在繁殖过程中能量变化特征及其分子机制。

3、为了探究热应激如何影响文蛤免疫系统对细菌感染的响应,我们在夏季 正常海水温度(27℃)和接近热应激温度(31℃)条件下,开展了文蛤副溶血弧 菌感染实验。分别设置了 27℃非感染组(27℃-control)、27℃弧菌感染组 (27℃-vibrio)、31℃非感染组(31℃-control)和 31℃弧菌感染组(31℃- vibrio) 等四组实验。结果表明,副溶血弧菌感染和温度都会影响死亡率,是否感染弧菌 决定了死亡事件的有无;而在感染的前提下,温度的高低又决定了死亡率的高低。 对免疫相关基因的表达模式进行 PcoA 分析,表明弧菌感染在潜伏期的影响起主 导的,随着感染的进程弧菌感染和温度的影响逐渐均衡。采用 TaqMan 技术进一 步分析发现,在适宜环境的个体(27℃-control)对肝胰腺中的弧菌清除的能力 显著高于其他组。我们进一步探究了温度和弧菌感染对凋亡和自噬的影响,结果 显示潜伏期(3d)温度和病原感染会使文蛤抗自噬和抗凋亡能力上调,而恢复期 (8d)温度升高和病原感染会降低其抗凋亡和抗自噬的水平。最后分析了温度和 弧菌感染对机体抗氧化酶活性的影响,结果显示,27℃-control 组中 MDA、GSH 和SOD 的含量显著低于其他 3 组,MDA 和 GSH 含量在 31℃-control、27℃-vibrio 之间没有差异,但显著高于 27℃-control,且显著低于 31℃-vibrio 组,表明单独 的温度或病原体刺激会对机体产生相似的氧化刺激,在两种因素的共同作用下, 会对机体产生显著叠加的应激作用。上述结果探究了水温和机体抗感染免疫的关 系,为解释贝类在夏季高温时期的规模性死亡提供了依据。

4、转录组数据提示 TLR 通路在繁殖前后发生变化,而 Toll-like 受体是经典 TLR 信号通路的关键分子,在免疫、生殖等方面具有重要意义。在本研究中, 我们鉴定了文蛤的 MpTLR 和 MpMyD88 的 ORF 序列,预测的 MpTLR 蛋白具有 N 端 EGF 结构域和 C 端 TIR 结构域;MpMyD88 具有 N 端 DEATH 结构域和 C 端 TIR 结构域。进化分析表明,文蛤与 Mizuhopecten yessoensis 和 Pecten maximus 聚为同一支,这暗示了 MpTLR 和其他贝类具有同源性。MpTLR 的组织分布分 析表明,MpTLR 在肝胰腺、闭壳肌、鳃、性腺和外套膜均有高表达。LPS 和 polyI:C 刺激后,MpTLR mRNA 表达显著上调,提示其参与了文蛤的免疫应答。此外, 敲低 MpTLR 后,MyD88,BCL2,MAPKK 和 IκB 的 mRNA 表达量显著降低, 而 TBK1 和 NF-κB 的 mRNA 表达量无明显变化,提示 MyD88,BCL2,MAPKK 和 IκB 是受到 MpTLR 调控的下游靶基因。最后,我们发现 MpTLR 可以调控 NF-κB 从细胞质转移到细胞核内。上述结果为进一步研究 TLR 通路在文蛤繁殖 和免疫中的功能提供了基础。

Other Abstract

Clam Meretrix petechialis is an important commercial mollusc in China. Due to the environmental stress and climate changes, the mass mortality often occurs in the culture process, which causes huge economic loss to aquaculture industry. Mass mortality of clams usually happens in the high temperature summer, meanwhile, summer is also the reproductive season for clams. Because animal reproduction and immune response to pathogens are processes of high energy demand, there may be a trade-off between reproduction and immunity. In this study, in view of the summer mortality of bivalve molluscs, transcriptome and proteomics analysis were performed on this clam before and after reproduction to reveal the effects of reproductive process on immunity ability and energy metabolism. Through multi-angle experiments, we also investigated the influences of high temperature and Vibrio pathogen on clam survival, immune function, oxidative stress and apoptosis autophagy. Based on the transcriptome, the TLR signaling pathways affected by reproduction were selected to explore its immune function and the molecular mechanism. The main results of this study are as follows: 1. In order to analyze the effect of reproductive process on the clam immunity, we selected clam samples before and after reproduction and performed transcriptome analysis. Based on the BGISEQ-500 sequencing platform, a total of 48339 unigenes were obtained. A total of 1805 differential genes (DEGs) were detected in the female comparison group (Pre_F-vs.-Post_F), while 2488 DEGs were detected from the male comparison group (Pre_M-vs.-Post_M). KEGG enrichment analysis of DEGs showed that immune signaling pathways common to both male and female groups changed after reproduction, including the IL-17 signaling pathway, NOD-like receptor signaling pathway, TLR signaling pathway, and RIG-I-like receptor signaling pathway, etc. Twenty-two immune-related DEGs were further screened by Nr annotations, which from complement and coagulation cascade pathway, apoptosis pathway, TLR signaling pathway, NF-κB signaling pathway, leukocyte transenverse migration pathway, MAPK signaling pathway, etc. Further analysis found that female up-regulated the expression of apoptosis, TLR signaling pathway, and heat shock-related genes post spawning, while male down-regulated the expression of complement-related genes. In addition, we conducted a challenge experiment on clams before and after spawning, and detected the expression of four immune-related genes (ARAF-like, CREM, TLR4 and TBK1). The results showed that the immune response to Vibrio stimulation was up-regulated in both male and female clams after reproduction. We also used two marker genes DOUX and MITF to evaluate the immune status of the clam before and after reproduction under Vibrio challenge. The transcriptional and protein levels of DOUX and MITF were significantly up-regulated in the post reproduction samples under the pathogen stimulation, which further confirmed that reproduction affected the clam immune response level to pathogen challenge. These results provide a new understanding about reproductive effects on immunity. 2. Since reproduction requires a large amount of energy, the impact of reproduction on metabolism is increasingly concerned by scientists. In this study, proteomics analysis was used to obtain hepatoproteomics from calms before and after reproduction. Analysis based on iTRAQ yielded a total of 5,192 proteins, of which 42 and 37 DEPs were detected in female and male, respectively. The function of the identified DEPs was explored through the KEGG pathway analysis, and the results showed reproduction has serious influences on metabolic processes. The expressions of four DEPs were decreased after reproduction, which is the glycogen debranching enzyme (AGL) in female, mannose-6-phosphate isomerase (MPI), β-glucuronidase (GUSB) and glyoxylate/hydroxypyruvate reductase (GRHPR) in male. It means that carbohydrate metabolism is weakened after reproduction. Additionally, the expressions of aldehyde dehydrogenase (NAD+) in female and meprin B and cysteine-S-conjugate β-lyase in male increased after reproduction, suggesting reproduction enhanced protein and amino acid metabolism. In addition, glutathione dehydrogenase/transferase (DHAR) and glutathione S-transferase (GST) were up-regulated in female and down-regulated in male, indicating that reproduction induces more severe oxidative damage in female than in male. The expressions of the troponin, myosin and EFHD2 in female were down-regulated after reproduction, indicating that reproduction may weaken the muscle function of female. Finally, total glucose and protein levels increased briefly after reproduction and then returned to pre-reproduction levels. Our results are helpful to better understand the molecular mechanisms of energy changes in the clams during reproduction. 3. In order to explore how temperature affect the clam immune system response to pathogen, we conducted Vibrio challenge experiment under normal temperature (27°C) and close to heat stress temperature (31°C) in summer. Four experimental groups were set up: 27°C non-infected group (27°C-control), the 27°C Vibrio infection group (27°C-vibrio), the 31°C non-infected group (31°C-control) and the 31°C Vibrio infection group (31°C-vibrio), respectively. The results showed that both Vibrio infection and temperature could affect clam mortality. Vibrio infection determined the death event, and the temperature determined the mortality rate in the case of pathogen infection. PcoA analysis of the immune-related gene expression patterns showed that the influence of Vibrio infection was dominant in the incubation period, and the effects of Vibrio infection and temperature gradually balanced with the progress of infection. Further analysis showed that clams in suitable environment (27°C-control) had significantly higher ability to eliminate Vibrio from hepatopancreas than in other groups. We explored the effect of temperature and Vibrio infection on clam apoptosis and autophagy, and the results indicated that temperature and pathogenic infection would increase anti-autophagy and anti-apoptotic ability in incubation period (3d), while they would reduce the anti-apoptosis and anti-autophagy level in recovery period (8d). Finally, we analyzed the effect of temperature and Vibrio infection on the activity of antioxidant enzymes. The results showed the contents of MDA, GSH and SOD in the 27℃-control group were significantly lower than those in the other three groups, MDA and GSH contents had no difference in 31°C-control and 27°C-vibrio group, but significantly higher than that of 27°C-control and significantly lower than that of the 31℃-vibrio group. The results indicated that temperature or pathogen alone could produce the similar oxidative stress, under the combined action of the two factors, they would have a significant additive effect to the clam. Our results explored the relationship among water temperature, pathogen and clam immunity, and provided reference for explaining the clam summer mortality. 4. Transcriptome data suggested TLR pathway changed before and after clam reproduction. Toll-like receptor is a key molecule of TLR signaling pathway and plays important roles in immune and reproduction. In this study, we identified the ORF sequences of MpTLR and MpMyD88 of this clam. The predicted MpTLR protein has an N-terminal EGF domain and C-terminal TIR domain, and MpMyD88 has an N-terminal DEATH domain and a C- terminal TIR domain. Phylogenetic analysis MpTLR showed it was clustered into the same branch as Mizuhopecten yessoensis and Pecten Maximus, suggesting homology between MpTLR and other molluscs. Tissue distribution analysis showed that MpTLR widely expressed in the hepatoscreas, muscle, gill, gonad and mantle. After stimulation with LPS and polyI:C, MpTLR mRNA expression was significantly up-regulated, suggesting it is involved in the immune response of clam. In addition, after knocking down MpTLR, the mRNA expression of MyD88, BCL2, MAPKK and IκB decreased significantly, while the expression of TBK1 and NF-κB did not change significantly, indicating MyD88, BCL2, MAPKK and IκB were downstream target genes regulated by MpTLR. Finally, we found MpTLR could regulate NF-κB transfer from the cytoplasm to the nucleus. These results provide clues for further study of TLR pathway in clam reproduction and immunity.

MOST Discipline Catalogue理学
Language中文
Table of Contents

第一章 引言..................................................................................................................... 1 1.1 文蛤生物学及养殖现状.......................................................................................... 1 1.2 文蛤的生殖................................................................................................................ 2 1.3 文蛤的免疫防御....................................................................................................... 3 1.3.1 贝类的病害............................................................................................................... 3 1.3.2 贝类免疫的特点......................................................................................................5 1.3.3 贝类主要的免疫通路.............................................................................................7 1.4 文蛤的代谢..............................................................................................................16 1.4.1 碳水化合物代谢....................................................................................................16 1.4.2 脂代谢...................................................................................................................... 17 1.4.3 蛋白质或氨基酸代谢.......................................................................................... 18 1.5 生殖、代谢和免疫的权衡....................................................................................18 1.5.1 生殖与免疫的权衡...............................................................................................19 1.5.2 生殖与代谢的权衡...............................................................................................19 1.5.3 温度对免疫的影响...............................................................................................20 1.6 本研究的内容和意义..................................................................................................21

第二章 生殖过程对文蛤免疫影响的转录组分析..........................................................22

2.1 研究背景.................................................................................................................. 22 2.2 材料方法.................................................................................................................. 23 2.2.1 实验动物..................................................................................................................23 2.2.2 RNA-Seq 和数据分析..........................................................................................23 2.2.3 差异表达基因(DEGs)分析...........................................................................24 2.2.4 弧菌攻毒和样本采集.......................................................................................... 24 2.2.5 qPCR 分析...............................................................................................................25 2.2.6 蛋白质印迹分析....................................................................................................26 2.3 结果........................................................................................................................... 27 2.3.1 序列组装和注释....................................................................................................27 2.3.2 差异基因(DEG)分析及免疫相关通路鉴定.....................................................28

2.3.3 生殖前后免疫相关基因表达差异................................................................... 31 2.3.4 弧菌感染下文蛤生殖前/生殖后基因表达变化..........................................33 2.3.5 弧菌感染下文蛤免疫状况评估........................................................................34 2.4 讨论........................................................................................................................... 35 第三章 生殖过程对文蛤代谢影响的蛋白组分析..........................................................38

3.1 研究背景................................................................................................................... 38 3.2 材料方法.................................................................................................................. 39 3.2.1 实验动物..................................................................................................................39 3.2.2 蛋白组测定.............................................................................................................39 3.2.3 蛋白定量和 iTRAQ 数据分析.......................................................................... 39 3.2.4 生物信息学分析....................................................................................................40 3.2.5 生殖恢复期葡萄糖和蛋白代谢的测定.......................................................... 40 3.3 结果........................................................................................................................... 40 3.3.1 生殖前后的蛋白质组.......................................................................................... 40 3.3.2 生殖影响的代谢相关 KEGG 通路.................................................................. 41 3.3.3 生殖后碳水化合物代谢减弱.............................................................................43 3.3.4 生殖后蛋白质和氨基酸代谢增强................................................................... 44 3.3.5 与肌肉运动和氧化应激相关的蛋白质的变化............................................ 44 3.3.6 葡萄糖和蛋白质含量在生殖过程中短暂上升后恢复到产前水平................... 45

3.4 讨论........................................................................................................................... 46 第四章 生殖季节高温和弧菌感染对文蛤免疫的影响................................................. 49

4.1 研究背景.................................................................................................................. 49 4.2 材料方法.................................................................................................................. 50 4.2.1 实验动物..................................................................................................................50 4.2.2 DNA 提取、RNA 提取和 cDNA 的获得......................................................51 4.2.3 TaqMan 检测肝胰腺组织中的载菌量............................................................51 4.2.4 qPCR 与 PCoA 分析............................................................................................ 52 4.2.5 蛋白印迹分析........................................................................................................ 53 4.2.6 氧化应激水平的检测.......................................................................................... 53 4.3 结果........................................................................................................................... 53 4.3.1 温度和弧菌共同影响文蛤的生存能力................................................................ 53

4.3.2 文蛤肝胰腺组织中的弧菌载菌量变化分析.................................................54 4.3.3 温度和弧菌影响条件下免疫基因表达模式的 PcoA 分析.......................55 4.3.4 温度和弧菌感染对凋亡和自噬的影响.......................................................... 57 4.3.5 温度和弧菌感染对机体氧化应激的影响.............................................................58

4.4 讨论........................................................................................................................... 59 第五章 文蛤受生殖影响的关键免疫通路 TLR 的研究................................................63

5.1 研究背景.................................................................................................................. 63 5.2 材料方法.................................................................................................................. 64 5.2.1 实验动物..................................................................................................................64 5.2.2 实验引物..................................................................................................................64 5.2.3 RNA 提取和 cDNA 的合成...............................................................................65 5.2.4 MpTLR-ORF 获得和序列分析.........................................................................65 5.2.5 MpTLR 对不同刺激物的响应实验................................................................66 5.2.6 qPCR.........................................................................................................................66 5.2.7 RNA 干扰实验.......................................................................................................66 5.2.8 蛋白印迹分析实验...............................................................................................67 5.2.9 酵母双杂实验........................................................................................................ 67 5.3 结果........................................................................................................................... 68 5.3.1 MpTLR 和 MpMyD88 基因序列分析............................................................ 68 5.3.2 MpTLR 组织分布................................................................................................. 70 5.3.3 MpTLR 对不同病原的响应分析......................................................................70 5.3.4 MpTLR 下游靶基因筛选................................................................................... 71 5.3.5 MpTLR 对 NF-κB 调控模式的研究................................................................73 5.3.6 体外 MpTLR 和 MpMyD88 无直接相互作用............................................. 74 5.4 讨论........................................................................................................................... 74 结 论.................................................................................................................................. 79 参考文献............................................................................................................................. 81 附 录................................................................................................................................111 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/337002/178322
Collection中国科学院海洋研究所
Recommended Citation
GB/T 7714
王迪. 生殖过程对文蛤免疫和代谢影响的研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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