温度对底播虾夷扇贝适合度性状影响的研究

IOCAS-IR > 实验海洋生物学重点实验室

	温度对底播虾夷扇贝适合度性状影响的研究
	刘超1,2
学位类型	博士
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	北京
学位专业	海洋生物学
关键词	底播虾夷扇贝适合度性状环境因子转录组学分析家系育种
摘要	虾夷扇贝（Patinopecten yessoensis）是一种重要的冷水性经济贝类之一。其自然分布区域位于西北太平洋，在韩国、日本以及中国均有大规模的人工养殖。目前虾夷扇贝的养殖方式主要包括浮筏养殖和底播养殖。底播养殖由于不需要额外的养殖设施，可以减少养殖成本获得更高的养殖效益。在中国，獐子岛海区是虾夷扇贝底播养殖的主要区域。2008年之前，长海县虾夷扇贝底播面积已经达到122万亩左右，如今长海县獐子岛虾夷扇贝的确权海域达到322万亩。但是随着底播面积的增加，许多新的问题不断出现，比如养殖个体小型化，回捕率降低等问题严重影响着獐子岛虾夷扇贝底播产业的发展。2011年之后，獐子岛虾夷扇贝的亩产一直稳定在80公斤左右，产量难以有大的提高。本研究以底播虾夷扇贝的标志物为抓手，研究底播虾夷扇贝季节性生理、营养指标及基因表达水平与生产性能的相关性，尝试构建现场及实验室水平的扇贝底播效率精确评估方法，探讨影响扇贝底播效率的关键环境因子；开展免疫学及转录组学研究，尝试探讨虾夷扇贝对高温及温度剧变等关键环境因子应答及分子机制；通过构建大规模家系，依据数量遗传模型，探讨关键胁迫环境因子（温度）下虾夷扇贝的适应潜力，获得适合底播养殖的虾夷扇贝品种。以期对虾夷扇贝的底播养殖提供有益的借鉴。 1. 底播虾夷扇贝生产性能同环境因子、抗氧化代谢酶活性以及相对表达量关系的模型 2012年12月-2014年12月期间同大连獐子岛集团股份有限公司合作，在獐子岛海洋牧场选择4个站位进行影响底播虾夷扇贝适合度性状环境因子的调查工作。通过调查，我们发现底播虾夷扇贝在每年的12月至次年6月的生长速度（壳长和重量）明显高于6月至12月的生长速度（每年下半年几乎停止生长）。通过线性回归分析发现海水温度、溶解氧含量、叶绿素含量、悬浮性颗粒物（SPM）以及颗粒性有机物 (POM) 等环境因子严重影响了底播虾夷扇贝的生长、存活、性腺指数、肥满度等生产性能。营养组分（糖原含量）以及几种重要的免疫代谢相关酶活性（谷丙转氨酶GPT、乳酸脱氢酶LDH、超氧化物歧化酶SOD以及总抗氧化能力TAOC）及其基因的相对表达量均会对虾夷扇贝的生产性能产生影响。通过以上调查分析，我们建议虾夷扇贝底播场地应该选择建在温度偏低的地区以降低死亡率提高性腺指数，选择悬浮性颗粒物（SPM）含量较低的地区，以提高壳长生长率、重量生长率以及性腺指数，选择颗粒性有机物（POM）含量较高的地区，以提高底播虾夷扇贝的肥满度。使用线性回归分析，获得了同虾夷扇贝壳长生长率、重量生长率、性腺指数、肥满度、死亡率具有显著相关性的几种重要的代谢以及抗氧化酶及基因表达量。基于这些酶活性以及基因相对表达量数据首次利用主成分分析的方法构建了预测底播虾夷扇贝生产性能的预测模型，经过Pearson关联分析可知，这些预测模型同相应的生产性能之间具有显著的相关性（P< 0.05）。这些模型是对虾夷扇贝生长率、死亡率传统调查方法的进一步补充，对底播虾夷扇贝养殖业的发展具有一定的促进作用。 2. 虾夷扇贝对高温刺激的分子应答机制在实验室条件下使用相应的酶活试剂盒以及RNAseq技术分别从生理、转录组学层面对高温导致虾夷扇贝死亡的因素进行探究。受到28℃高温刺激以及恢复过程中，二龄贝的死亡率显著高于一龄贝。Cox模型分析结果显示，高温刺激后虾夷扇贝的存活率同贝龄、GPT活性、p53含量、TAOC活性以及HSP70含量变化均显著相关（P < 0.05），其中GPT活性同扇贝的存活负相关（B < 0），其余因素同扇贝存活呈显著正相关（B > 0）。此外，高温导致虾夷扇贝心跳紊乱以及心输出量的减少，进而减少血淋巴对机体的供氧能力，导致扇贝死亡。通过转录组学分析可知，在恢复24h时一龄贝中ATP相关基因表达量下调导致虾夷扇贝死亡；二龄贝中ATP合成过程以及ATP合成过程中电子传递过程导致了ATP合成量降低更严重，导致了扇贝死亡率的升高。而且二龄贝在恢复24h时错配修复相关基因也显著下调，导致DNA错配概率升高，在受到高温刺激之后导致其死亡。再者，一龄贝在整个恢复过程中核糖体合成相关基因表达量的上升，在一定程度上减少了虾夷扇贝的死亡，同时二龄贝在恢复48h条件下才能出现核糖体合成以及翻译相关基因的显著上升表达，说明受到高温刺激之后的恢复能力方面二龄贝明显不如一龄贝。受到高温刺激之后，相对于二龄贝HSP70等相关基因在一龄贝体内表达量显著升高，也在一定程度上解释了二龄贝死亡率偏高的问题。 3. 虾夷扇贝底播养殖性状的遗传评估利用獐子岛集团2011年和1985年自日本引进的虾夷扇贝为亲本，通过家系育种方法于2013年2月培育73个家系，在2013年12选择35个家系进行虾夷扇贝标记底播实验，在2014年12进行统计计算其死亡率并同浮筏养殖的同一家系进行比较，得到10个底播存活率显著高于浮筏养殖的家系。同时使用AI-REML（或Quasi-REML）方法对2013年家系进行遗传参数评估。依据遗传参数计算每个个体及家系的估计育种值（EBV），依据30%：70%经济加权后计算各个家系及个体的综合育种值。共筛选21个家系作为选育F2家系的亲本。在2015年利用筛选出的家系进行杂交，利用底播以及浮筏养殖个体作为对照构建2015年家系。选育组及对照组按常规沉箱及浮筏养殖流程分6个测定点进行培育至2016年12月，目前该部分数据正在采集中。
其他摘要	The yesso scallop Patinopecten yessoensis is a cold-water species distributed in the northwest Pacific Ocean and is cultured commercially in Japan, Korea, and China using suspended longline aquaculture and bottom sowing culture. Bottom sowing culture can achieve higher breeding efficiency because of no need of additional breeding buoyancy. The sea raching around Zhangzidao Island is the most important area for yesso scallop bottom sowing culture. In 2008, the bottom sowing area of yesso scallops has reached 80 thousand hectares, now the permitted area of sea ranching reached 3.22 million mu. But with the development of bottom sowing culture, some new problems, such as lower ratio of recapture and smaller individual harvest sizes, have constantly emerged and seriously inhabited the bottom sowing industry development in Zhangzidao island. Since 2011, the shell yield has been stable at around 80 kg/mu and difficult to be improved. In this study, we focused on the correlation among seasonal variation of physiological, nutritional component and relative gene expression level to performance traits, trying to construct the accurate method for assessing bottom sowing efficiency on field and laboratory levels and explore the key environmental factors affecting bottom sowing aquaculture of yesso scallops. Depending on the immunology and transcriptomics research, we try to illuminate the yesso scallops’ response to heat shock and corresponding molecular mechanism. At last, a large scale yesso scallops families were instructed to select the species suitable for being bottom sown with the help of simulation experiments of bottom sowing aquaculture. We believe our research will provide useful references for the yesso scallop bottom sowing industry. 1. The correlation models among performance traits and environmental factors, immunity and metabolism enzymes activities and corresponding relative expression From December 2012 to December 2014, the investigation was conducted in 4 sitese located in yesso scallops’ bottom sowing ranching with the help of No. 19 research vessel affiliate to Zhangzidao Group Co., Ltd.. Through the investigation, we found bottom sowed yesso scallops’ growth rate (shell length and total tissue weight） from December to the next June was significantly higher than that from June to December. The linear regression analysis showed that environmental factors such as temperature, dissolved oxygen content (DO), chlorophyll content, suspended particulate matter(SPM) and particulate organic matter (POM) seriously affect the bottom sowed yesso scallops’ performance traits such as growth rate (shell length and total tissue weight), mortality rate, gonad index (GI), condition factor (CF). Biochemical composition (glycogen content), metabolism and antioxidant enzymes [glutamic-pyruvic transaminase (GPT), lactate dehydrogenase (LDH), superoxide dismutase enzyme (SOD) and total antioxidant capacity (TAOC)] and their relative expression also influenced the bottom sowed yesso scallops’ performance traits significantly. Through the above investigation and analysis, we suggest bottom sowing sites should be chosen in the area of low temperature to reduce mortality and improve the gonad index (GI), in the area of lower content of suspended particulate matter (SPM) region to increase growth rate (shell length and total tissue weight) and the gonad index (GI) and in the area of higher particulate organic matter (POM) content to improve the bottom sowed yesso scallops’ condition factor (CF). The immunity and metabolism enzymes and corresponding relative expression significantly related to performance traits were selected using linear regression analysis. The principal component analysis (PCA) were used to construct the models predicting the bottom sowed scallops’ performance traits based on the analysis of linear regression analysis. The models were further supplement for the traditional survey methods of yesso scallops’ performance traits. They also played a certain role in promoting the development of shellfish aquaculture. According to Pearson correlation analysis, the correlation between prediction models and the corresponding performance traits were significantly (P <0.05). 2. Yesso scallops’ response to heat shock and corresponding molecular mechanism In laboratory, the enzyme activity kits and RNAseq technology were used to explore the factors that cause the mortality of the scallops suffering from heat shock on the level of physiology and transcriptomics. The Cox model analysis showed that mortality resulting from heat shock was related to shellfish age, GPT activity, p53 content, TAOC activity and HSP70 content significantly (P < 0.05). GPT activity was negatively related to scallop mortality (B<0), while the other factors with scallop mortality was significantly positive correlation (B > 0). Besides, heat shock resulted in the reduction of heart rate and cardiac output, which implies the decreasion of oxygen supply capacity of the hemolymph for tissues, which resulted in the mortality of scallops. The transcriptome analysis showed that, the down-regulation of gene expression related to ATP synthesis resulted in the mortality of one-year elder yesso scallops after recovering for 24h. The reasons for the two-year elder scallops included the following several factors. The gene related to ATP synthesis, the electron transfer and mismatch repair were down-regulated, which leaded to reducing the amount of ATP and increasing the amount of mismatched gene. Besides, during the recovery the one-year elder scallops’ gene related to ribosome synthesis were up regulated. This up-regulation perhaps reduced the one-year elder scallops mortality in a certain extent. These upper reasons explained the reasons for lower mortality of one-year elder scallops than that of two-year elder. For two-year elder scallops, the up-regulation of gene related to ribosome synthesis appeared in the period after recovering for 48h, which will be one evidence for better recovery ability from heat shock in one-year elder scallops than two-year elder scallops. 3. The genetics estimates of traits for bottom sowed yesso scallops In February 2013, 73 families of yesso scallops were bred in Zhangzidao group Co., Ltd., 35 families of these were chosen to be utilized in marked bottom sowing experiment. The comparation between the the same family’s survive of bottom sowed aquaculture and suspending longline aquaculture. The results showed that 10 families’s survival rates were significantly higher in the s marked bottom sowed experiment. The genetic parameter estimation were processed with the help of AI-REML or Quasi-REML method. The estimated breeding values (EBV) of every family and individual were calculated basing on the genetic parameter, while the comprehensive EBV were also calculated basing on the 30%:70% economic weight. At last, 21 families were chose as the parent of F2 bred in 2015. In 2015, 78 families were bred as F2 and 23 families were bred as control falimies with the parents chosen from bottom and suapending aquaculture industry of Zhangzidao Group Co., Ltd. The following exprements were conducted.
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/337002/112525
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实验海洋生物学重点实验室 2.中国科学院大学
第一作者单位	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	刘超. 温度对底播虾夷扇贝适合度性状影响的研究[D]. 北京. 中国科学院大学,2016.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
温度对底播虾夷扇贝适合度性状影响的研究.（5648KB）	学位论文		限制开放	CC BY-NC-SA	浏览