以斑马鱼为模式研究脂肪酸受体CD36在鱼类脂味觉感知中的作用

Taste acts as the last step for an animal's food acceptance or rejection behavior by evaluating the nutritious content and the overall pleasure of the food. Recently more and more evidences raise the possibility for the taste system in the role of the perception of lipids in mammals. Also, the fatty acid receptor CD36 has been proved to be as an important candidate of fat taste receptor. Fish has different taste modality with mammals, however our work has suggested that fish can also perceive to fatty acids. CD36 is highly conserved during evolution and is widely present in the vertebrates, but it is no report about the function of CD36 in fish taste. We has shown that CD36 is highly expressed in the taste buds in fish, and the expression of CD36 is regulated upon the linoleic acid stimulants, indicating the role of CD36 in fat perception in fish. Based on these, the zebrafish is used as a model in this project, and we would further investigate the location of CD36 in fish taste bud cell by detecting the co-expression with the marker molecular of taste bud cell; then detect the response of zebrafish CD36 to fatty acid and the signal pathway mediated by CD36. Also, we would further uncover the role of CD36 as a receptor in transferring the fat taste signal by calcium imaging technology; and detect the change of the sensitivity to fatty acid after CD36 knock out by TALENs or Cas9 technology in zebrefish. This project would benefit to understand the molecular mechanism of fish fat taste perception and the evolution of taste. It has also the potential instruction to the fish nutrition and diet development.

哺乳动物脂味觉及其受体CD36的确定是近年来激动人心的新发现，而对鱼类味觉的研究目前还相当薄弱。我们的前期研究表明：虽然与哺乳动物有很大的不同，但鱼类也具有脂味觉，且CD36在鱼类味蕾呈显著表达，并受脂肪酸调控。故CD36作为鱼类候选脂味受体很值得期待。本项目拟以斑马鱼为材料：1）通过检测CD36基因/蛋白与味觉细胞标记分子的共表达，确认其在味觉细胞的定位；2）结合体内体外实验，深入分析脂味物质对CD36基因/蛋白表达的特异调控，并揭示其是否介导脂味觉信号途径；3）运用钙离子成像技术探究CD36在脂味觉感知中作为受体的功能；4）观察基因敲除前后斑马鱼摄食脂味饵料能力的变化，综合分析CD36在鱼类脂味觉感知中的作用。本研究将为阐明鱼类脂味觉在摄食选择和感知过程中的分子机制奠定基础，深化对动物味觉进化的认识，并可对水产实践中鱼类饵料的开发提供理论依据。

味觉系统在哺乳动物摄取脂类的感知中扮演着重要角色。鱼类的味觉虽然与哺乳动物有很大的不同，但我们的前期研究发现鱼类也具有脂味觉，但还不清楚其脂味觉受体是什么。本项目以斑马鱼为模式，从斑马鱼CD36的蛋白三维结构、亚细胞定位、组织表达特异性、亚油酸诱导表达特征、细胞水平实验及在体行为学实验等多角度证明斑马鱼cd36在脂味觉感知中发挥重要作用。主要结果为：1）斑马鱼CD36预测的三维结构为发卡式膜结构，从结构上支持其作为受体的角色。2）通过原位杂交和免疫组化技术证实CD36基因与蛋白定位于斑马鱼的味蕾，从组织定位上支持其在味觉感知中的作用。3）证实亚油酸（LA）可以调控zCD36的表达，且zCD36特异地介导了亚油酸引起的脂味觉信号途径。4）通过TALEN技术获得了cd36基因敲除的斑马鱼，并通过行为学实验证明了zCD36作为鱼类脂味觉受体的功能。本研究首次鉴定出CD36为鱼类的候选脂味觉受体。另外，本项目还发现斑马鱼cd36基因也在脂类代谢调控与免疫防御中发挥作用，从脂味觉-脂质代谢-免疫防御的逻辑链条拓展了对CD36功能的认识。总之，本研究为阐明鱼类脂味觉感受的分子机制奠定了基础，深化了我们对动物味觉进化的认识，且可为水产实践中鱼类营养与饵料的开发提供科学依据。