许氏平鮋妊娠生理特征及代谢组学对温度胁迫的响应机制

The significant changes or stress of ocean temperature have an adverse effect on gonad development and reproductive process of fish, and this leads to the sharp decrease of offshore Sebastes schlegeli population. It was speculated that water temperature stress from sea ice may influence the reproduction activity and the recruitment of offshore rockfish population. However, there are not systematic reports about the physiological adaptation and evolutionary mechanism of ovoviviparous fish during pregnancy. Gas Chromatography-Mass Spectroscopy (GC-MS) method, molecular biology and endocrine techniques were used to clarify the physiology characteristics of Sebastes schlegeli during gestation period (fertilization, fertilized egg development, larva development, parturition) by studying the reproductive cycle of wild Sebastes schlegeli in this research. The effects of temperature stress on gonad metabolomics pathway were observed and we analysed the content change of related gene and hormone. Related gene expression and hormone marker detection methods which obtained in our laboratory were used to verify and determine some major candidate genes and sex hormones playing key roles in the pregnancy of Sebastes schlegeli, and then pregnancy molecular marker detection method was established. These studies clarified pregnancy metabolomics physiological mechanism of ovoviviparous fish in theory, laid the foundation for revealing ovoviviparous fish reproductive evolutionary strategy and provided the scientific basis for resources protection and severe environmental factors assessment of offshore rockfish in practice.

海水温度剧烈波动（温度胁迫）可对鱼类性腺发育和生殖过程产生不良影响，近年来北部海区出现的海冰导致的温度胁迫可能影响了卵胎生许氏平鮋的生殖活动及其种群数量补充，而关于卵胎生鱼类妊娠生理适应与进化机制的系统研究尚未见报道。本课题采用气相色谱-质谱联用（GC-MS）、基因和蛋白表达、内分泌学等检查方法，通过研究野生许氏平鮋生殖周期，查明其妊娠期（受精、受精卵发育、仔鱼发育、分娩）生理特征；观测温度胁迫对实验鱼血清代谢组学的影响，分析它们在此过程中变化规律；采用本实验室建立的基因与蛋白检测技术进行代谢通路寻找和功能验证，结合代谢组数据确定许氏平鮋妊娠期发挥主效作用的候选基因，建立妊娠检测代谢物标记。通过这些研究，在理论上阐明卵胎生鱼类妊娠生理的代谢组学机制，为揭示鱼类生殖进化策略奠定基础，在实践上为近海岩礁鱼类资源保护与灾害性环境因子评估提供科学依据.

鱼类属变温动物，它的生长繁衍与温度、光照、紫外、气压等气候因子有密切关系，其中温度是鱼类生长发育最重要的气候因子。水温影响着鱼类的摄食、生长、免疫和生殖等活动，具有多方面生态作用。许氏平鲉是我国北方沿海人工养殖的重要品种之一。其生存温度范围较广（5～28℃），生长适宜温度18-24℃。目前在全球变暖的大背景下，黄海北部的海域水温波动剧烈且昼夜温差急剧扩大，对网箱养殖许氏平鲉造成严重的影响。本课题以许氏平鲉为对象，采用生理学，内分泌学，分子生物学，转录组学及代谢组学等研究方法，对关键基因、关键代谢产物及关键通路进行筛选，在不同层面上对许氏平鲉响应急性温度胁迫的生理学应答机制进行研究。2015年活国家自然科学基金课题资助，研究了许氏平鲉妊娠生理特征及响应温度胁迫的分子机制。主要包括对热低氧诱导因子HIF-αs基因的克隆和表达分析；急性高温胁迫下雄性许氏平鲉血液生理及包括hsp70在内的热相关基因的表达分析；侧重研究了急性温度胁迫对许氏平鲉内分泌机能的影响，肝脏代谢机能和血液指标的影响，以及急性温度胁迫下肝脏转录组及血清代谢组学的分析；同时还对卵胎生许氏平鲉胚胎离体培养、发育形态学和早期性腺分化进行了研究。在理论上丰富许氏平鮋分子生理机制，为揭示卵胎生鱼类生殖策略提供科学依据；在实践上为掌握许氏平鮋繁殖规律及建立全人工繁殖技术奠定基础。研究结果研究学术论文7篇，其中3篇论文发表在SCI期刊上，培养博士和硕士研究生7名，先后有4次在国内相关学术会议上进行交流。