实验海洋生物学重点实验室知识库

牡蛎是营固着生活的双壳贝类，多分布于亚热带与温海地区，在中国渤海、黄海以至南沙群岛等都有出产，且品类繁多，是人类可利用的重要海洋生物资源。在牡蛎经济性状相关研究中，肥满度（条件指数）是其中一个重要方向。在牡蛎养殖过程中经常出现肥满度剧烈波动情况，造成无法准确把握采收时期，从而对养殖企业的生产造成影响。本研究选取长牡蛎作为实验对象，采用人工震荡和海区观测等研究方法，结合生物信息学和分子生物学分析方法，在生理生化以及基因表达水平，研究三倍体牡蛎肥满度剧烈变化的原因。

牡蛎的生长依赖于它们与环境之间的稳定关系，牡蛎对外部环境的依赖性使其极易受到环境波动的影响，高密度笼养或吊养等养殖方式均会造成牡蛎个体因海浪冲击受到长期的晃动或震荡胁迫，一些海区牡蛎肥满度的快速变化常常发生在大型风浪前后，据此推测肥满度变化可能与养殖模式造成的震荡胁迫有关。为探究震荡胁迫对肥满度变化的影响，在2022年9月份“梅花”台风过后初步研究了山东胶南养殖海区距离台风路径不同距离位点长牡蛎的肥满度数据，结果表明台风影响大的海区牡蛎肥满度降低。通过人工模拟海浪震荡处理长牡蛎，发现SOD、CAT、糖原、T-AOC等生理指标发生变化。进一步通过非靶向代谢组学方法研究了单体长牡蛎在模拟风浪震荡胁迫条件下的代谢活动差异。将单体牡蛎放入风浪震荡模拟装置中进行6小时的震荡冲击处理，分别采集未受震荡个体、震荡后0小时、6小时和18小时个体的血浆质谱数据，共筛选出447种共同差异代谢物。通过对各组样品间差异代谢物进行KEGG代谢通路富集分析发现，震荡应激主要影响了甘油磷脂代谢、鞘脂代谢、亚油酸代谢和花生四烯酸代谢等脂质代谢途径的变化。与甘油酯水解相关的产物变化显著，表明牡蛎受到海浪胁迫应激后产生了一系列应激反应和病理生理过程。

三倍体牡蛎性腺发育及风浪造成的生殖活动可能也是造成肥满度变化的影响因素。本研究在同一养殖群体中选取不同性腺发育状态的三倍体牡蛎进行组织学和转录组学比较。对性腺石蜡切片的分析发现三倍体与二倍体相比，生殖细胞数量减少、败育和异常聚集，但是怀卵量存在显著的个体间差异。差异表达基因（DEGs）的GO和KEGG通路富集分析结果表明，三倍体牡蛎性腺发育状态与DNA复制、微管运动、细胞核分裂和核酸切除修复等相关。选取其中6个基因进行Realtime qRT-PCR验证，结果与转录组数据一致。三倍体牡蛎可能通过突破这些基因表达调控的限制实现了生殖细胞的产生，当性腺发育到成熟阶段，依然容易受到海浪等外部条件的影响，产生生殖行为，从而导致肥满度的变化。

Oysters, a group of sessile bivalve mollusks predominantly inhabiting subtropical and temperate seas, thrives in the coastal waters of China. They are widely cultured because of the important economic values. Among the various economic traits of oysters, plumpness or condition index, stands as a pivotal parameter. Fluctuations of plumpness pose challenges for precise harvest timing, impacting aquaculture management. In this study, the causes behind significant plumpness variations in triploid oysters was investigated with approaches that integrate physiological, biochemical, RNA sequencing, alongside bioinformatics, morphological, and cytological techniques.

Oysters' growth is inherently linked to environmental stability. Their sensitivity to environmental fluctuations renders them susceptible in fluctuating conditions. High-density farming practices, such as caging or suspension, expose them to long-term wave-induced agitation, which precedes notable plumpness shifts, particularly around severe weather events. This study posits that oscillatory stress-induced cultivation conditions may govern fecundity fluctuations by influencing the metabolism or spawning. In order to investigate the effect of shock stress on the change of plumpness, a field study was preliminarily conducted on the plumpness of oysters post the pass of typhoon Muifa in Jiaonan, Shandong in September 2022. The results revealed distinct plumpness correlating with the degree of typhoon impact on the sampled sites at varying distances from the storm's trajectory. Laboratory-induced shaking experiments further illustrated alterations in antioxidant enzymes, energy reserves, and overall antioxidative capacity.

An untargeted metabolomics study was further employed to oysters under simulated wind and wave stress. Singleton oysters were placed into a wave shock simulator for 6 h of shock treatment, and serum mass spectrometry data were collected from unshocked individuals, and individuals at 0 h, 6 h and 18 h after shock, respectively. A total of 447 differential metabolites were identified. KEGG metabolic pathway enrichment analysis of the differential metabolites revealed that shaking stress predominantly disrupted lipid metabolism, implicating pathways like glycerophospholipid and sphingolipid metabolism, linoleic acid and arachidonic acid metabolism, with implications for stress responses and physiological dysfunction.

Gonad development and subsequent spawning induced by shake stress may also contribute to the plumpness variation. Transcriptomic profiling were conducted on selectively cultured triploid and diploid oyster, differing in gonadal maturity. Genes implicated in DNA replication, microtubule dynamics, nuclear division, and DNA repair were enriched in the differentially expressed genes (DEGs), reinforcing the hypothesis that disrupted germ cell proliferation underlies reduced fecundity in triploids. Validation via RT-PCR corroborated transcriptomic findings, while histological assessments highlighted diminished germ cell counts, abortions, and irregular aggregations in triploids compared to diploids.

In conclusion, this study illuminates how mechanical stress influence the lipid metabolism. Specific gene expressions may play roles in overcoming the fecundity limitations in triploid oysters, enabling them to achieve substantial gonadal development. These insights contribute to a more nuanced understanding of the complex interplay between genetics, environment, and physiology, with potential implications for optimizing oyster aquaculture practices.

第1章 绪论... 1

1.1  牡蛎养殖现状... 1

1.2  条件指数与肥满度... 1

1.3  长牡蛎的生长与环境因素的关系... 2

1.4  牡蛎性别决定机制... 2

1.5  牡蛎性腺发育... 3

1.6  多倍体的发展现状... 3

1.7  三倍体长牡蛎的养殖现状... 4

1.8  代谢组学在水产养殖的应用... 5

1.9  研究的计划及意义... 6

第2章 实验材料与方法... 9

2.1  实验材料... 9

2.1.1  实验动物与材料... 9

2.1.2  研究所用主要耗材和试剂... 9

2.1.3  仪器设备... 11

2.2  实验方法... 11

2.2.1  “梅花”台风过境实海观测... 11

2.2.2  实验室内震荡模拟及其生理指标测定... 12

2.2.3  过氧化氢酶（CAT）含量的测定... 12

2.2.4  糖原含量的测定... 12

2.2.5  总蛋白含量的测定... 13

2.2.6  丙酮酸激酶（PK）活力的测定... 13

2.2.7  果糖磷酸激酶活性（PFK）的测定... 14

2.2.8  丙二醛（MDA）含量的测定... 14

2.2.9  总抗氧化能力（T-AOC）活力的测定... 15

2.2.10  SOD活力的测定... 15

2.3  基因表达测定... 16

2.3.1  总RNA提取... 16

2.3.2  反转录... 16

2.3.3  实时荧光定量聚合酶链反应... 16

2.3.4  数据分析... 17

2.4  性腺切片制作... 17

2.4.1  固定液配制... 17

2.4.2  性腺石蜡切片的制作... 17

2.5  代谢组学分析... 18

2.5.1  海浪对单体牡蛎物质代谢的胁迫机制研究... 18

2.5.2  代谢物提取... 18

2.5.3  液相色谱质谱分析... 18

2.5.4  数据预处理... 19

2.5.5  数据分析... 19

2.6  转录组学分析... 20

第3章 实验结果... 23

3.1  台风过后胶南海域笼养长牡蛎肥满度调查... 23

3.2  风浪震荡对牡蛎生理活动的影响... 24

3.2.1  震荡后牡蛎生理指标检测... 24

3.2.2  代谢组学分析结果... 25

3.3  三倍体牡蛎性腺发育分析及机制解析... 29

3.3.1  三倍体牡蛎外部形态描述... 29

3.3.2  三倍体牡蛎性腺的组织学分析... 29

3.3.3  转录组学分析... 31

第4章 讨论... 39

4.1 风浪对牡蛎代谢的影响... 39

4.1.1 甘油磷脂代谢... 39

4.1.2 鞘脂代谢... 39

4.2  三倍体性腺发育机制... 41

4.2.1 DNA复制... 41

4.2.2 核酸切除修复NER机制... 43

4.3 研究的不足之处以及展望... 45

第5章 结论... 47

参考文献... 49

致  谢... 57

作者简历及攻读学位期间发表的学术论文与其他相关学术成果    59