长牡蛎对海洋酸化的响应与适应机制研究

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	长牡蛎对海洋酸化的响应与适应机制研究
	王秀丹
学位类型	博士
导师	宋林生
	2017-05-10
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	长牡蛎海洋酸化 Co2 碳酸酐酶游离的腺苷酸环化酶转录组
其他摘要	海洋酸化（ocean acidification, OA）对海洋生物，尤其是海洋钙化生物有潜在的负面影响。其直接影响是对生物体内酸碱平衡的干扰，并进一步影响钙化过程、能量代谢和免疫反应等。长牡蛎（Crassostrea gigas）是生活在河口和潮间带区域重要的双壳贝类，具有重要的生态地位和经济价值。本研究综合运用分子生物学、生物信息学、生理学、免疫学及转录组学等相关技术，查明了几种关键酸碱调节蛋白的结构特征及其在长牡蛎对酸化响应过程中的生理调节功能，分析了长牡蛎血淋巴细胞在短期和长期CO₂胁迫下的基因表达变化，初步探讨了双壳贝类对海洋酸化的响应和适应机制。本研究从长牡蛎基因组中克隆得到一个细胞内碳酸酐酶（CgCAII-1）。通过生物信息学分析，CgCAII-1在进化中相对保守，含有一个典型的CA结构域，不含跨膜域或信号肽。重组蛋白rCgCAII-1能将CO₂转化为H⁺和HCO₃^-，受到CA抑制剂acetazolamide（AZ）的抑制。酸化处理后，其在闭壳肌、外套膜、肝胰腺、鳃、血淋巴细胞中的表达量显著增加，且CgCAII-1蛋白定位在血淋巴细胞膜上，且主要聚集在外套膜上皮细胞、膜泡结构以及鳃丝内部参与化学平衡的维持。酸化处理实验以及酸化和抑制剂共处理实验表明，高浓度CO₂使得血淋巴细胞内的pH值（intracullular pH, pHi）显著上升且碳酸酐酶参与调控pHi。酸化处理后，鳃和外套膜中的碳酸酐酶活性下降或保持不变，表明长牡蛎在CO₂胁迫下可能通过提高CgCAII-1的表达，减弱其对CO₂的转化并加强其与膜蛋白等的作用，从而促进离子运输和酸碱调节。本研究克隆获得长牡蛎分泌型碳酸酐酶（CgCA）的cDNA序列，发现其含有一个信号肽和一个典型的CA结构域。通过对其发育时期和组织表达水平检测，发现其在D-型幼虫时期表达水平最高，且主要分布于外套膜和血淋巴细胞中，表明其可能参与钙化过程。酸化处理后，CgCA在闭壳肌、外套膜、肝胰腺、鳃和血淋巴细胞中的表达水平显著上调，且其蛋白形式能够在血淋巴细胞的细胞质中和细胞膜上以及外套膜的褶皱结构中发生聚集，说明其可能聚集到长牡蛎的钙化结构中。同时，酸化处理还使得血淋巴细胞内Ca²⁺浓度显著降低。进一步的抑制处理和CgCA干扰实验发现，CgCA与血淋巴细胞的Ca²⁺浓度负相关。钙离子试剂Ionomycin处理实验发现，细胞内Ca²⁺浓度的变化影响CgCA在外套膜组织中的分布。据此，本研究提出CgCA-Ca²⁺互调机制，酸化处理下这种互调作用可能更为密切且影响长牡蛎钙化平衡的维持和贝壳的分泌。本研究从长牡蛎基因组中鉴定出了游离的腺苷酸环化酶（soluble adenylyl cyclase, sAC）的同源基因CgsAC并克隆表达了其CycC结构域。酶活测定实验发现，重组蛋白MBP-rCgsAC和鳃组织中的天然蛋白均受HCO₃^-激活，生成cAMP，且受到sAC特异性抑制剂KH7的抑制。酸化处理后，CgsAC在闭壳肌、外套膜、肝胰腺、鳃和血淋巴细胞中的表达水平显著上调。免疫印迹实验发现其在组织中蛋白发生剪切作用生成45 kD和20 kD的蛋白。同时，血淋巴细胞内部分CgsAC从细胞质中转移到细胞膜上。酸化处理后，鳃和外套膜组织中cAMP含量及血淋巴细胞的pH值显著升高，且这些变化受到KH7的抑制，说明酸化处理激活了HCO₃^-/CgsAC/cAMP信号通路。酸化处理实验以及酸化和KH7共处理实验表明，高浓度CO₂引起的血淋巴细胞的吞噬率的下降、ROS水平的上升、凋亡率的下降、组织和线粒体ATP的变化等过程均受到CgsAC的调节。血淋巴细胞caspase-3和caspase-9活性测定实验发现，caspase-3和caspase-9活性线性相关，而caspase-8活性没有变化。表明酸化能够激活线粒体凋亡途径，且CgsAC参与调控由酸化引起的凋亡。本研究共构建了正常组、短期组（CO₂处理7天）、长期组（CO₂处理60天）共9个长牡蛎血淋巴细胞转录组文库，共得到72,504,095单端变长的reads，共鉴定出28,027个基因，其中短期组/正常组之间共有501个差异基因，长期组/正常组之间共有2728个差异基因。对差异表达基因中FPKM值倍数变化< 0.5或> 2的基因进行了GO富集分析，发现在短期组和长期组上调表达基因中，细胞成分类别中注释为‘membrane’及分子功能（MF）类别中注释为‘ion binding’的基因比例较大。分子功能类别中注释为‘heterocyclic compound binding’的基因占有较大比例，大多数基因与ATP、GTP的结合或DNA、RNA的结合相关，与短期组相比，长期组中更多与运输和信号转导的基因和DNA、RNA结合相关的基因上调表达。长期组差异表达基因中，分子功能类别中注释为‘Calcium ion binding’的基因也占有较大比例，且主要为上调表达。此外，细胞内的Ca²⁺水平短期酸化处理下显著下降，长期酸化处理下恢复到正常水平，而血清中钙含量持续升高，说明长牡蛎可能通过钙结合蛋白将细胞内Ca²⁺转运到血清中从而实现细胞内Ca²⁺的充分补偿。血清中的葡萄糖短期酸化处理下显著下降，而长期的酸化处理下显著上升，显著高于对照组。肝糖原的变化趋势与葡萄糖的变化趋势类似。因此，短期的酸化处理会引起膜蛋白和离子结合相关基因、ATP或GTP结合相关的基因（如细胞骨架基因）的差异表达，造成细胞内钙离子水平的上升和血清中葡萄糖含量下降，同时储存的肝糖原加速分解等应激反应。而长牡蛎在长期的酸化处理下，通过激活更多离子和酸碱调节蛋白的转录作用，诱导与复制、转录、翻译等过程相关的基因的表达，上调表达大量钙结合蛋白实现细胞内Ca²⁺的充分补偿，增加营养物质的吸收和储存等多种调节机制，以适应高浓度CO₂环境。综上所述，面对不断上升的CO₂浓度和下降的pH，长牡蛎已建立了自身的应对策略。其能迅速的增加CA的表达水平，促进CA的功能转变，加强对酸碱平衡和钙化水平等的调节，激活HCO₃^-作为信号分子的HCO₃^-/CgsAC/cAMP调节通路，调节下游免疫反应和能量代谢等过程。另外，长牡蛎作为典型的潮间带生物，其已建立了短期酸化处理下的应激和长期酸化处理下的适应机制。 ; Ocean acidification (OA) has been demonstrated to have severe effects on marine organisms, especially marine calcifiers. Pacific oyster (Crassostrea gigas) is an important marine calcifier living in the estuaries and intertidal zones and also one of dominant aquaculture bivalves worldwide. In the present study, the molecular characteristics, expression profile and phylogeny relationship of three key enzymes, as well as their roles in regulation of physiological processes in response to OA were explored. The transcriptome libraries of haemocytes from the oyster C. gigas after short-time and long-time CO₂ exposure were sequenced in order to find new clues of the mechanism of mollusk response or adaptation to OA. CgCAII-1 possessed a conserved CA catalytic domain, with high similarity to invertebrate cytoplasmic or mitochondrial α-CAs. Recombinant CgCAII-1 could convert CO₂ to HCO₃^-, which could be inhibited by AZ. The mRNA transcripts of CgCAII-1 in muscle, mantle, hepatopancreas, gill and haemocytes increased significantly after exposed to elevated CO₂. CgCAII-1 could interact with the haemocytes membrane proteins and the distribution of CgCAII-1 protein became more concentrated and dense in gill and mantle under CO₂ exposure. The intracellular pH (pHi) of haemocytes under CO₂ exposure increased significantly (p < 0.05) and CA inhibition reduced the pHi value. Besides, there was no increase in CA activity in gill and mantle after CO₂ exposure. The impact of CO₂ exposure on CA activity coupled with the mRNA expression level and protein translocation of CgCAII-1 provided evidences that CgCAII-1 could respond to ocean acidification and participate in acid-base regulation. The cDNA of CgCA was of 927 bp encoding a predicted polypeptide of 308 amino acids with a signal peptide and a CA catalytic function domain. The mRNA transcripts of CgCA were constitutively expressed in all tested tissues with the highest levels in mantle and hemocytes. During the early development period, the mRNA transcripts of CgCA could be detected in all the stages with the highest level in D-veliger larvae. Elevated CO₂ increased the mRNA transcripts of CgCA in muscle, mantle, hepatopancreas, gill and hemocytes significantly (p < 0.05) and induced the translocation of CgCA in hemocytes and mantle. Moreover, elevated CO₂ also caused the decrease of intracellular Ca²⁺ in hemocytes (p < 0.05). The inhibition of CA by acetazolamide and suppression of CgCA gene via RNA interference could increase the intracellular Ca²⁺ in hemocytes (p < 0.05). Besides, the decrease of intracellular Ca²⁺ content caused by Ca²⁺ reagent ionomycin could affect localization of CgCA in mantle tissue. The results indicated CgCA played essential roles in calcification and elevated CO₂ accelerated the mutual modulation between calcium and CgCA, implying reduced calcification rate and dissolved shells under OA. Soluble adenylyl cyclase (sAC) is an acid-base sensor in response to HCO₃^- and an intracellular source of cyclic AMP (cAMP). In the present study, an ortholog of sAC was identified (designated as CgsAC) and the catalytic region of CgsAC was cloned and expressed. Similar to the native CgsAC from gill tissues, the recombinant CgsAC protein (rCgsAC) exhibited HCO₃^- mediated cAMP-forming activity, which could be inhibited by a small molecule KH7. After 16 days’ CO₂ exposure treatment (pH=7.50), the mRNA transcripts of CgsAC increased in muscle, mantle, hepatopancreas, gill, male gonad and haemocytes, and two truncated CgsAC forms of 45 kD and 20 kD were produced. Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO₂ exposure. Besides, CO₂ exposure could increase the production of cAMP and intracellular pH of haemocytes, which was regulated by CgsAC (p < 0.05), suggesting the existence of a HCO₃^-/CgsAC/cAMP signal pathway in oyster. The elevated CO₂ could induce the increase of ROS level (p < 0.05) and decrease of haemocytes phagocytic rate (p < 0.05), which could be inhibited by KH7. The apoptosis ratio in CO₂ exposure group (35.2 %) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO₂ could be significantly inhibited (p < 0.05) by KH7. After CO₂ exposure, CgsAC was found to be co-localized with mitochondria in the cytoplasm, and the pro-caspase-3 was cleaved into two small fragments. Moreover, the activities of caspase-3 and caspase-9 also increased post CO₂ exposure and these increases could be inhibited by KH7. However, the activities of caspase-8 did not change significantly compared with that in the control group. After CO₂ exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could be also be inhibited by KH7. While the ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO₂ exposure, which was also inhibited by KH7. The results collectively suggested that CgsAC was an important acid-base sensor in oyster and the HCO₃^-/CgsAC/cAMP signal pathway might be responsible for intracellular alkalization effects on oxidative phosphorylation and innate immunity under CO₂exposure. The transcriptome libraries of haemocytes from the oyster C. gigas after 7-day and 60-day CO₂ exposure were sequenced. Nine double-end fragment libraries were constructed and sequenced in the three groups, including blank, short and long group, and there were 72,504,095 single-end reads totally obtained with 28,027 genes identified. There were 501, and 2728 differentially expressed genes identified in the comparison of short versus blank, and long versus blank, respectively. Genes in the ‘membrane’ and ‘ion binding’ were enriched in the ontology of ‘cellular components’ in both the short versus blank and long versus group and more genes related to transport were up-regulated in the long group than the short group. A set of significantly differently expressed genes belonging to the category ‘molecular function’ were enriched in ‘heterocyclic compound binding’. In the short versus blank genes list, these genes were mainly ATP or GTP binding, such as cytoskeleton genes including actin, tubulin and dynein. In the long versus blank genes list, there were more genes involved in ATP or GTP binding and DNA or RNA binding including cytoskeleton genes and replication, transcription and translation related genes. ‘Calcium ion binding’ is another category highlighted by the GO enrichment analysis and varieties of calcium binding proteins (CBP) were up-regulated in the long group. Intracellular calcium concentration decreased significantly after short time CO₂exposure and then increased to the level of the control group after long time exposure, while calcium content in the serum increased in both short and long group. Besides, the energy metabolism of the short and long groups were different. In the short group, both the glucose in serum and liver glycogen content decreased significantly, while in the long group the glucose in serum and liver glycogen content, were much higher than that of the blank group. The present results indicated that oyster displayed different mechanism in short-time CO₂ stress response and long-time CO₂ adaptation. In conclusion, key acid-base sensors including CA and sAC mediated acid-base regulation and downstream immune response and energy metabolism in oyster under CO₂ exposure. As important marine calcifier, oyster displayed different mechanisms in response and adaptation to elevated CO₂.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136569
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	王秀丹. 长牡蛎对海洋酸化的响应与适应机制研究[D]. 北京. 中国科学院大学,2017.

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