海洋酸化对泥蚶受精动力学的影响与机理研究

1/3-1/4 of the anthropogenic CO2 is absorbed by the ocean, which leads to significant decrease of the seawater PH (Ocean Acidification) and subsequently affects marine species especially marine shellfishes. Since ocean acidification is a research topic with only about 10 years' history, many research works are needed to be carried out including the effect and mechanism of ocean acidification on fertilization kinetics of marine mudflat shellfish blood clam. Blood clam, Tegillarca granosa, is one of the typical mudflat shellfishes that live in costal muddy substances. In one hand, the coastal mudflat where blood clam live is facing the most severe anthropogenic CO2 release. In the other hand, the spawning season of blood clam, which is in summer, overlaps with CO2 temporal distribution peak. Therefore, blood clam are under tremendous ocean acidification threaten. Preliminary study showed lab ocean acidification simulation by both CO2 bubbling and pH adjusting by HCl led to significant lower fertilization success of blood clam, no matter which type of gametes was exposed to acidified treatment, and the fertilization rates were only about 14-39% of that of control. In addition, preliminary gene ontology analysis showed that the differentially expressed genes of blood clam between ocean acidification treatment and control were mainly located on plasma membrane (about 50%) and associated with transmembrane transportation biological process (about 27.3%). Therefore, whether ocean acidification affects fertilization success of blood clam through its influences on fertilization kinetics and the membrane proteins such as gamete reorganization and ion channel proteins is an interesting question. This study will focus on how and why does ocean acidification affect the first and important step of reproduction, fertilization, of blood clam. Both the effects of ocean acidification on each fertilization kinetics parameter and on the overall fertilization kinetics model will be explored. Moreover, the mechanism of these effects will be further studied through investigating the membrane proteins such as gamete reorganization and ion channel proteins of the gametes. The results of this work will help to expend the scope of ocean acidification research, to fill the gaps in affecting mechanism of ocean acidification on fertilization, and to provide the experimental data for aquaculture industry to choose strategy dealing with coming ocean acidification.

1/3至1/4的人源CO2排放将会被海洋吸收，从而导致海水pH值显著下降，对海洋生物特别是海洋贝类产生严重影响。作为一个近十年刚刚发展起来的研究领域，海洋酸化对滩涂贝类受精动力学影响及机理的研究尚属空白，亟待开展。前期预实验发现泥蚶配子的酸化处理使其受精率下降为对照组的14-39%。而且基因注释分析显示酸化组所差异表达的基因主要是位于质膜上（大约50%）参与跨膜转运的基因（大约27.3%）。本研究将以泥蚶为研究对象，研究海洋酸化对泥蚶繁衍重要环节受精过程的影响，探讨海洋酸化对泥蚶受精动力学重要参数的作用，研究海洋酸化如何整体地影响泥蚶受精动力学模型，并从配子膜蛋白（离子通道、配子识别蛋白）水平分析其作用机理。本研究的结果将有助于丰富海洋酸化研究内容，弥补海洋酸化对海洋生物受精影响机理的研究空白，为海洋贝类养殖产业在未来如何应对海洋酸化提供基础实验参考。

1/3至1/4的人源CO2排放被海洋吸收，从而导致海水pH值显著下降，对海洋生物特别是海洋贝类产生严重影响。但是到目前为止，海洋酸化对滩涂贝类受精动力学影响及其机理的研究尚属空白。针对此科学问题，本课题在定量分析海洋酸化对泥蚶受精作用的基础上，通过模型构建、组学分析等技术方法，从精卵碰撞概率、精卵相互识别以及钙波形成多个方面综合解析了海洋酸化影响泥蚶受精的作用机理。研究结果表明，不论采用二氧化碳充气还是添加无机酸、以及不论是精子还是卵子进行海洋酸化处理都导致泥蚶受精率显著下降。同时泥蚶精子的运动能力相较于H+浓度更易受到pCO2变化的影响。通过构建受精动力学模型，对比分析了海洋酸化对泥蚶精卵碰撞概率与相互识别的影响，发现海洋酸化显著降低了泥蚶的碰撞概率与精卵识别成功率。通过细胞内钙离子荧光染色技术，本研究还发现海洋酸化显著抑制了受精卵的钙波形成，使其细胞内钙离子浓度的峰值与持续时间均出现显著下降。通过对泥蚶卵细胞膜蛋白进行蛋白质组分析，本研究筛选得到了共10个与受精相关的蛋白，并通过抗体封闭的方法证明了卵膜钙网蛋白在精卵识别中具有重要功能。进一步的基因表达分析表明海洋酸化显著抑制了卵膜钙网蛋白与钙离子通道基因的表达，可能是海洋酸化导致精卵识别与钙波形成障碍的原因。综上所述，本研究发现海洋酸化会降低精卵碰撞概率，干扰精卵识别，阻碍钙波的正常形成，从而综合性的导致受精率的显著下降。本课题获得的研究结果将有助于丰富海洋酸化研究内容，为海洋贝类养殖产业在未来如何应对海洋酸化提供基础实验参考。

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