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皱纹盘鲍度夏死亡与高温胁迫响应的分子机制研究
林思恒1,2
学位类型硕士
导师张国范
2016-05-21
学位授予单位中国科学院大学
学位授予地点青岛
学位专业水产养殖
关键词皱纹盘鲍 度夏死亡 生理变化 高温胁迫 心率测定
摘要皱纹盘鲍是我国重要的海水养殖贝类,近年来福建等南方海区已成为其主养区。但作为引种自北方的物种,南方皱纹盘鲍养殖产业经常出现夏季大规模死亡问题,困扰着产业的发展。初步研究表明,高温胁迫可能是引起鲍夏季大规模死亡的主要原因之一。针对这一问题本研究对皱纹盘鲍的度夏死亡展开生物学调查,并进行高温胁迫响应的分子机制初步研究。
本研究首次对我国福建宁德三沙湾养殖的二龄皱纹盘鲍进行一系列生物学指标的检测,包括死亡率、性腺发育情况和各项生理指标,同时记录养殖环境的环境数据。性腺指标的测定是通过检测每月性腺的面积大小计算。结果表明:度夏期间出现了较高的死亡率,且死亡率随温度上升不断上升,直至温度下降后,死亡率的增加才变缓,度夏期间总死亡率达到了58.58%。与此同时,鲍鱼的性腺发育程度并没有明显的差异,说明性成熟并不是导致夏季的死亡的主要原因。分析生理指标的测定结果,发现这些指标与环境压力密切相关,尤其是度夏期间的高温和不稳定的溶解氧含量。能量代谢指标中蛋白质、糖原、游离脂肪酸、乳酸脱氢酶均有明显的月度变化,所有的能量指标都呈减少趋势;抗氧化能力指标包括超氧化物歧化酶和总抗氧化能力都在度夏期间先上升后下降。这些变化说明各类供能物质含量下降,机体在度夏过程中的能量大量消耗并影响正常的能量分配;机体无法保证免疫活动所需能量,导致免疫能力下降,减弱的抗氧化能力无法抵御过氧化损伤,可能导致了死亡。虽然结论未有直接证据解释皱纹盘鲍的夏季大规模死亡,但这些结果将会对研究皱纹盘鲍高温胁迫响应的分子机制提供有效信息,也有助于解决养殖过程出现的问题,改善和提高度夏成活率。
对贝类进行高温胁迫下生存能力的鉴定也是研究生理响应及高温适应机制等科学问题的重要环节。本研究应用一种非损伤性的心率检测方法,检测两个皱纹盘鲍养殖群体在高温胁迫条件下心率等生理指标的变化,以心率变化指标精确并快速比较这两个群体高温耐受能力。由于高温胁迫下皱纹盘鲍的心率随温度变化的关系符合阿伦尼乌斯公式,且心率随温度上升呈先上升后下降,该研究通过计算两者直线拟合拐点即阿伦尼乌斯拐点温度(ABT,Arrhenius break temperatures)指标,用以指示皱纹盘鲍温度耐受程度。以此法对高温耐性皱纹盘鲍群体和对照群体各17个个体进行了测定分析,并记录每个个体的生长指标。结果表明:两个群体间的ABT存在显著差异,高温耐性组的皱纹盘鲍的ABT显著高于对照组(P<0.05);个体ABT指标的高低与测定个体的壳高正相关(P<0.05)。本研究首次探讨了高温胁迫下皱纹盘鲍心率变化规律,并以ABT指标分析比较了两个皱纹盘鲍养殖群体间高温耐受能力。
为了解释皱纹盘鲍高温胁迫响应的分子机制,我们对高温耐性和高温敏感的两个群体进行了转录组测序和不同胁迫处理的表达谱测序。转录组测序组装获得了96,803条Unigene,组装完整性较高。对Unigene进行功能注释,包括与Nr、Swiss-Prot、KEGG、COG、GO和Pfam数据库的比对,共获得26,006条注释结果。差异表达基因初步结果为:高温胁迫前后差异表达基因7,912个,高温耐性群体与高温敏感群体间差异表达基因4,933个,一龄个体与二龄个体间差异表达基因6,463个。不同的高温耐受程度、高温胁迫与否以及实验个体的年龄都是导致基因差异表达的原因。这些结果为将来的高温胁迫响应相关功能基因的确定和分子标记育种提供基础信息。
其他摘要Pacific abalone, Haliotis discus hannai, has become the predominantly farmed abalone species in Fujian, southern China since 2006. As an introduced abalone species from northern China, mass mortality of the farmed Pacific abalones often occurred during summer days in Fujian in recent years. In order to better understand the phenomenon of summer mortality in the farmed Pacific abalones in southern China, we would make the thorough investigation about this phenomenon and elucidate preliminarily the molecular mechanism of resistance to heat stress in Pacific abalone.
In this study, we aimed to provide data in terms of summer mortality occurrence and management in this abalone species reared in sea-based farming system of southern China. For the first time, we conducted a field investigation to monitor biological condition of the two-year-old animals including the monthly survivals and visual gonad index in Sansha Bay, Fujian, a major Pacific abalone farming site from April to October 2014. We also measured the monthly variation in metabolic parameters and antioxidative capacities in the same experimental animals. A range of environmental parameters were assessed and correlated with biological indicators for abalone condition, metabolic parameters and antioxidative capacities. Results showed that a total of 58.58% mortality rate finally occurred after summer days in the sea-based farmed abalones according to the filed investigation. Visual gonad index assessment showed that the sampled animals had similar gonad ripeness levels during each sampling date. Environmental parameters and the abalone mortalities monitored in the study showed significant positive correlation between seawater temperature and the mortality of abalones. The metabolic parameters include protein, glycogen, nonesterified fatty acid (NEFA) content, and the enzyme activities of lactate dehydrogenase (LDH) and glutamic pyruvic transaminase (GPT) were analyzed. Analysis showed all the metabolic parameters, except enzyme activity of GPT, had significant variations during the sampling times. Despite the different levels of variation in each parameter in varying tissues, all energy resources showed a decrease tendency after summer. The analysis of antioxidative capacities included superoxide dismutase (SOD) and total antioxidative capacity (T-AOC). Results showed that both of these two parameters increased in the first few months and then decreased in September and October subsequently. The variations of the metabolic parameters and antioxidative capacities detected indicated that the insufficient energy supply and the peroxidative damage happened in the farmed animals during summer days. These findings might guide further molecular genetics-based studies that focus on the above-mentioned two aspects to elucidate the physiological processes underlying summer mortality in Pacific abalone and are applicable to the management and development of Pacific abalone aquaculture in sea-based farms of southern China.
Determination of the survival ability in abalones under heat stress is an important part in this study. We can get the survival indicator of Pacific abalones by measuring the heart rate of abalones. Heart rate were measured using the non-invasive method then get the Arrhenius break temperatures as the biomarker that determine the heat resistance of abalones with higher accuracy and faster way. Heart rate of abalones would rise as temperature increases then drop abruptly. The temperature that induce the cardiac failure in abalone is the Arrhenius break temperature (ABT). In this study, we measured the heart rate in 17 resistant abalones and 17 contrast abalones that from two different groups. The result illustrated that the ABT of resistant abalones seemed higher (P<0.05) than that of contrast abalones. Significant (P<0.05) positive correlation was found between shell height of abalone and ABT. In the first time, we determine the heat resistance of Pacific abalones by measuring the heart rate and its feasibility was verified.
Based on resistant and susceptive families as experimental materials, we applied transcriptome profiling analysis to investigate the molecular mechanism of resistance to heat shock stress in Pacific abalone respectively. There were 96,803 unigenes produced in total with high integrity. And the result of annotation showed that 26,006 unigenes were annotated in Nr, Swiss-Prot, KEGG, COG, GO, Pfam database. We screened differentially expressed genes (DEGs) between different treatment groups. We founded 7,912 DEGs between before and after heat stress groups; 4,933 DEGs between susceptibility group and resistance group; 6,463 DEGs between one-year-old and two-year-old abalones groups. Different treatment include different resistances to heat stress, before and after heat stress and different ages would result in genes expressed differentially. These finding could be helpful in future selection of Pacific abalone with enhanced resistance to heat stress.
学科领域水产养殖学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/112511
专题实验海洋生物学重点实验室
作者单位1.中国科学院海洋研究所
2.中国科学院大学
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林思恒. 皱纹盘鲍度夏死亡与高温胁迫响应的分子机制研究[D]. 青岛. 中国科学院大学,2016.
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