【作者】 柳意樊；

【导师】 李军；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 水产养殖， 2014， 博士

【摘要】 温度是影响鱼类存活、摄食、生长、发育、繁殖等过程的重要环境因子。鱼类对温度的耐受能力决定了其时空分布范围。随着全球气候变化和极端气象的频发，水生生物的耐热性研究及耐热品种的培育具有非常重要的研究意义和生产价值。然而，有关海水鱼类的耐热性研究还十分有限，耐热性指标也尚无统一的可靠标准。褐牙鲆（Paralichthys olivaceus Temminck&Schlegel，1846）是我国重要的海水养殖鱼类，具有较高的经济价值，养殖区域也不断南移。但受到夏季高温期死亡频发的影响，其养殖产业屡受损失。大西洋牙鲆（Paralichthys dentatusL.，1766）是大西洋海岸的重要渔获种，具有更好的高温耐受性。两者的杂交子一代P. olivaceus×P. dentatus （以下简称杂交鲆）继承了亲本优良的经济性状，同时表现出生长快、耐高温的特性。为了比较高温对褐牙鲆和杂交鲆的影响，阐明杂交鲆优于亲本的耐热性状，同时丰富海水鱼类耐热性研究，充实种间杂交遗传理论，为相关耐热性育种研究提供一定的理论基础，我们从组织水平、生理生化和hsp70基因表达量的水平研究了高温对褐牙鲆各个组织的结构、超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）、溶菌酶（LZM）、丙酮酸激酶（PK）的相对活力（相对含量）和hsp70基因表达量的影响。研究了不同升温模式下，褐牙鲆和杂交鲆鳃组织结构的变化，SOD、过氧化氢酶（CAT）、GSH、LZM和PK相对活力的变化，以及hsp70基因表达量的变化差异。同时还克隆、比较了褐牙鲆、大西洋牙鲆、杂交鲆hsp70/hsp90基因的cDNA序列，对29℃下三者的SOD、GSH、LZM和PK相对活力，以及hsp70/hsc70/hsp90基因的相对表达量变化进行了比较。主要研究结果如下：1)褐牙鲆不同组织对高温（32℃）的响应程度不同。肝、肾和鳃组织的结构在高温下出现了一系列的变化，如组织水肿、上皮位移或破裂、细胞坏死等。与氧化应激、免疫反应和能量代谢相关的酶活力检测发现，褐牙鲆的肝组织中SOD、GSH和LZM都有相对较高的表达。高温热胁迫下，不同组织的hsp70表达量随着时间的推移都表现出相似的变化趋势：先升高后下降。我们发现hsp70表达量达到峰值的时间大致有两个：2h和6h，并依据这个特点，把研究的组织分为两组，2h组包括脾、肝、鳃和肾，6h组包括肠、胃、肌、心和脑。通过各个组织hsp70表达量差异的横向比较，发现对照组中脑和鳃是hsp70表达较丰富的组织，高温胁迫条件下，肝、鳃和肌是hsp70表达量升高最显著的组织，分别对应1h、2h和6h（及12h）。值得注意的是，在组织学研究中表现出最多症状的组织（肝、肾和鳃）都在2h达到hsp70表达量的峰值，其中的肝和鳃的hsp70表达量变化尤为显著，且肝组织中酶的相对活力变化也较显著。由此我们提出，鳃和肝是研究高温对海水鱼类影响时理想的备选组织，相关的结论也为后续的研究工作提供了理论依据。2)慢性升温模式下，褐牙鲆在29℃和32℃时累计存活率显著低于杂交鲆；其鳃组织在高温下破坏严重；与氧化应激、免疫反应及能量代谢相关的酶（SOD、CAT、LZM和PK）的相对活力波动剧烈，在29℃和32℃附近基本都发生下降；hsp70基因表达量也在29℃和32℃时显著升高，且上调幅度显著高于杂交鲆，与这两个温度下的死亡爆发和组织损伤相对应。我们的研究发现，褐牙鲆对29℃和32℃的高温更敏感。相比之下，杂交鲆的累计存活率更高，在高温胁迫下其鳃组织的结构也相对保持得更完整，酶的相对活力变化不显著，hsp70的表达上调得更晚，上调幅度相对低于褐牙鲆，且在29℃后有适应后回落的趋势。这些结果都反应了其对高温可能具有更好的耐受性。至此我们总结并提出，高温不耐受种对高温的敏感性在多个层面都有所表现，如高死亡率、组织结构的严重破坏、酶活力的剧烈变化、hsp70基因的首先响应和大幅上调。与之对应的就是耐高温种在升温过程中于各个层次表现出的相对优势和稳定性。3)通过不同温度梯度（26、29和32℃）的急性升温模式实验我们发现：杂交鲆的累计存活率均高于褐牙鲆，在极端高温32℃下其死亡的发生（4h）和死亡的爆发（12h）均晚于褐牙鲆（1.5h，4h）；其鳃的结构相对保持得更完整，而褐牙鲆鳃的呼吸表面的破坏更严重；SOD、GSH、LZM和PK相对活力的变化基本反映了褐牙鲆和杂交鲆在极端高温（32℃）下，抗氧化能力和机体的免疫防御能力下降，同时伴随着能量支出的增加；两者的hsp70表达量在热刺激后0.5h就检测到显著升高，相比之下，褐牙鲆hsp70基因的表达量变化比杂交鲆更剧烈和持久，具体表现在更快的hsp70响应速度和更高的响应强度，以及更慢的回落速度。我们发现，急性升温模式和慢性升温模式所得结果具有一致性，反应了杂交鲆更好的耐热性，但两者相比，在慢性升温模式下杂交鲆的耐热性优势更明显。4)通过克隆褐牙鲆、大西洋牙鲆和杂交鲆hsp70和hsp90基因cDNA全序列及序列分析发现，三者的hsp70/hsp90基因高度保守，克隆所得的hsp90序列更接近hsp90-β。通过29℃热胁迫条件下，褐牙鲆、大西洋牙鲆和杂交鲆有关氧化应激、免疫反应和能量代谢的酶的相对活力研究和分子水平的研究发现：随着热胁迫时间的增长，SOD、GSH、LZM和PK的相对活力都发生了一系列的变化，且三者的变化趋势大致相同。尤其在SOD、LZM中的变化趋势较明显，变化曲线呈“M”型，类似于慢性升温模式下SOD和LZM的变化趋势。褐牙鲆、大西洋牙鲆和杂交鲆的hsp70/hsc70/hsp90基因表达量随着热胁迫时间的增长都发生了变化，hsp70基因表达量先升高后下降，而hsc70/hsp90基因表达量变化曲线呈“M”型，且hsc70/hsp90基因表达量变化的剧烈程度明显低于hsp70。结合前文慢性升温模式和急性升温模式的研究结论，我们认为hsp70是海水鱼类耐热性研究中较理想的指标。更多还原

【Abstract】 Temperature is regarded as an important abiotic variable influencing almost theentire life history of fish, including survival, ingestion, growth, development andreproduction. Temperature tolerance of fish limits its distribution, both in time andspace. As global climate change has increased extreme weather events, the study ofthermal tolerance mechanism and heat-tolerance breeding of aquatic organismbecomes a work of great value both in scientific research and aquaculture industry.However, works in thermal tolerance of marine fish are limited, and there lacksevaluation indicators of heat tolerance. Paralichthys olivaceus (Temminck&Schlegel,1846) is an ecologically and economically valuable flatfish species in China, whoseculture is widely distributed along the coast of North China, and is expanded to theSouth China. However, the aquaculture of this species has been suffered from the hotsummer, and is frequently shocked by mass mortality. Paralichthys dentatus (L.,1766)is a high temperature resistant species, which is also an important commercial flatfishalong the Atlantic coast. The hybrids of these two species (P. olivaceus×P.dentatus) inherits the excellent economic characters and performs well both ingrowth and thermal tolerance. We investigated the tissue specific responses in thehistological alteration, enzymic changes (superoxidase dismutase, SOD; catalase,CAT; reduced glutathione, GSH; lysozyme, LZM and pyruvate kinase, PK) and theexpression of hsp70of P. olivaceus to elevated temperature. We compared these twospecies under different heat modes. We also cloned the hsp70/hsp90genes of P.olivaceus, P. dentatus and the hybrids. Furthermore, we investigated the the changesof P. olivaceus, P. dentatus and the hybrids in related enzymes and hsp70/hsc70/hsp90expressions at29℃, with the purpose of clarifying the effect of elevated temperature to these species, which may provide us a better understanding of themechanism of heat tolerance and hybrid vigor in marine fishes. The main results areas follows:1) The tissues of P. olivaceus responded differently to the elevated temperature.There were a range of histological changes in liver, kidney and gill, including theedema, epithelial lifting, desquamation and necrosis. The SOD, GSH and LZMshowed relatively higher activity in liver. According to the time point of highestexpression of hsp70, the tissues could be divided into two groups: the2-h groupcomprised the spleen, liver, gill and kidney, while the6-h group comprised thegut, stomach, muscle, heart and brain. When investigated the tissue specificexpression of hsp70at respective time points, we found that the brain and gillwere tissues of rich hsp70expression before heat shock. However, the liver, gilland muscle had much higher hsp70expression under high temperature, with thetop expression at the time point of1h,2h, and6h (and12h).It was worth mentioning that, the tissues with the most significant lesions (gill,liver and kidney) also responded much earlier (2h) in hsp70expression than othertissues, especially the gill and liver, since they have shown the most outstandingmanifestations both in histological, enzymic and mRNA levels, which made themideal candidate tissues in the further thermal study of this important species.2) In chronic heat mode, the cumulative survival rate (CSR) of P. olivaceus wasmuch lower than that of the hybrids, with the gill structure damaged moreseriously. In P. olivaceus, the activity of SOD, CAT, LZM and PK fluctuateddramatically, all of which declined around the temperature of29℃and32℃.Moreover, the hsp70expression in P. olivaceus also increased significantly at29℃and32℃, whose up-regulated ranges were higher than those in the hybrids.All of these corresponded to the changes of CSR, which suggested the P.olivaceus was more sensitive to the temperature of29℃and32℃.However, in the hybrids, the CSR was higher, the gill structure under hightemperature was relatively more complete, the enzymic changes were more stable,and the response of hsp70was later than that in P. olivaceus, with a lower up-regulation and a trend of fall back above29℃. These all implied us the betterthermal tolerance of this species.So far, we could synthesize the characters of heat sensitive species, which mightbe revealed in many levels, such as lower CSR, severe damages in tissuesstructure, tremendous changes in enzymic activity and the earlier and strongerreaction in hsp70expression. In contrast, a thermal tolerance species alwaysshows better CSR and stability in other levels.3) In acute heat mode (26℃,29℃and32℃), the CSR in the hybrids was higher,with the lag of death occurrence (4h) and mass mortality (12h) compared to P.olivaceus (1.5h,4h), and the gill structure in the hybrids was more completed.The changes in enzymic activity reflected that, the oxidation resistance andimmune defenses were weakened by the extreme temperature both in P. olivaceusand the hybrids, with the increase of energy expenditure. The hsp70expressionincreased significantly at0.5h both in these two species. However, these reactionsof hsp70in P. olivaceus were more violent and persistent, which expressed infaster and higher response, as well as slower fall after rise. We found the resultsin acute heat mode were largely consistent with those in chronic heat mode,which suggested the hybrids had a better thermal tolerance. However, thisadvantage was more remarkable in the chronic mode.4) The full-length cDNA of hsp70and hsp90were cloned from P. olivaceus, P.dentatus and the hybrids. The analysis indicated these two genes were both highlyconserved. The hsp90isolated in our study was more closed to hsp90-β.When been heat shocked at29℃, the changes in enzymic activity (SOD, GSH,LZM and PK) showed relatively similar trends, especially in the SOD and LZM,whose curve turned out to be an “M”. Analogous curve has been found in chronicheat mode. The expression of hsp70got more tremendous and regular changesthan hsc70and hsp90, which made it an ideal candidate gene in the study ofthermal tolerance in marine fish.更多还原