海洋酸化影响长牡蛎左壳附着的生物矿化机制

Ocean acidification is the name given to the ongoing decrease in the pH and increase in acidity of the Earth's oceans,caused by the increasing uptake of the anthropogenically produced carbon dioxide, which would induce the negative even fatal physiological effects on marine organisms, and ultimately inpact the marine ecosystem. Oysters as the world wide distribution of shellfish (great masses of oysters can grow together to form oyster reefs) play an important role in the marine ecological system. As have been reported, the settlement of the oyster larva and the biomineralization process of many marine species can be disturbed by the acidification of the ocean. Settlement is also one kind of biomineralization. The left but not the right valve of the shell can undergo the attachment,so it is inferred that some specific functional proteins in the left valve must fulfill the biomineralization job during the important settlement phase in larvae stage. We can predict that the attachment of the valve would be interupted when those functional protein genes were interfered through immersing larva in dsRNA solution, probably by ocean acidification. Works will be launched to identify the proteins specific to settlement spot and test if these proteins would probably affect the settlement of the oyster. More information about the relationship of larva settlement frequency with the genes expression level，allele frequency or the related proteins activity will be acquired in the marine water or mineralization in vitro with different pH value, which would ultimately give us a glimpse into biomineralization mechanism of ocean acidification affecting larval settlement through left shell.

海洋酸化是指随着二氧化碳排放量的增加,海水的pH值逐渐下降的现象,而海水酸度的增加,会扰乱海洋生物的正常生理活动,甚至死亡,从而导致海洋生态环境的失衡。牡蛎作为世界上广泛分布的贝类，且可聚居成为牡蛎礁，在海洋生态系统中占据比较重要的地位。多篇文献已经报道，海洋酸化可降低贝类的附着率，也会干扰多种生物的矿化过程，而牡蛎附着也是一种生物矿化过程，并且只有左壳可以附着而右壳不能，那么左壳中应该存在一些特殊的蛋白来执行附着这项特殊的矿化功能，我们可以设想，如果这些特殊蛋白的生理功能受到海洋酸化的干扰，牡蛎的附着就不能完成，这可能也是海洋酸化影响牡蛎附着的原因之一。因此，本研究计划首先筛选到附着斑特有蛋白，然后，通过浸泡式RNAi验证相应基因对于幼虫附着的影响，最后,在不同pH值的水体中检验这些基因的表达、等位基因频率及其蛋白功能的变化，以此来探讨海洋酸化影响牡蛎幼虫左壳附着的生物矿化机制。

牡蛎作为世界上广泛分布的贝类，且可聚居成为牡蛎礁，在海洋生态系统中占据比较重要的地位。多篇文献已经报道，海洋酸化可降低贝类的附着率，也会干扰多种生物的矿化过程，而牡蛎附着也是一种生物矿化过程，并且只有左壳可以附着而右壳不能，那么左壳与右壳的差异蛋白或者基因，会与左壳附着及其对海洋酸化的响应有关。. 我们分析比较了长牡蛎左右外套膜间的差异表达基因、甲基化差异基因、差异蛋白、差异表达小RNA及其靶基因，并分析这些差异基因与长牡蛎左右壳间的形态和功能差异之间的关系，通过取交集，筛选出与长牡蛎附着有关的基因；通过荧光定量的方法检测EGF基因在幼虫附着变态前后的mRNA表达变化；对长牡蛎眼点幼虫开展海水酸化暴露试验，收集对照组及海水酸化暴露组的长牡蛎幼虫，对EGF基因的mRNA表达和等位基因频率的变化进行测定；对长牡蛎眼点幼虫开展添加EGF蛋白的海水酸化暴露试验，解释了海水酸化暴露对EGF蛋白调控长牡蛎矿化的影响情况；研究钾离子和两种钾离子通道抑制剂对太平洋牡蛎幼虫的附着与变态作用的影响。. 通过分析筛选出了长牡蛎左与右外套膜间的差异表达基因和甲基化差异基因；完成了长牡蛎左与右外套膜组织间的差异表达蛋白的比较分析和左右外套膜组织间小RNA的比较分析；通过比较分析，认为长牡蛎左壳附着可能与TGF-β信号通路有密切关系；但RNAi实验没有证实筛选出的相关基因对海洋酸化产生响应，需要进一步研究；通过研究两种钾离子通道抑制剂-----TEA（门控电压钾离子通道抑制剂）和格列本脲（内向整流性钾离子通道抑制剂）-----对牡蛎幼虫附着的影响，我们发现门控电压钾离子通道在长牡蛎幼虫附着中可能起着更重要的作用。. 长牡蛎左、右外套膜的转录组、甲基化组、蛋白组、小RNA组等数据，为左右对称动物对称器官的功能分化研究奠定了基础，也为长牡蛎附着及其对海洋酸化的响应研究提供了参考。