黑点青鳉Rh蛋白（Rhesus protein）家族功能分化及其机制研究

鱼类是唯一将氨氮作为氮代谢排泄产物的脊椎动物，具有独特的氨氮代谢生理和较高的氨氮耐受性。Rh蛋白家族是由多个进化相关的成员组成、具有氨氮转运活性的跨膜蛋白家族，在鱼类氨氮转运与排泄生理中发挥了重要作用。但目前该家族成员的功能特征、功能分化以及分化机制仍然未知。本项目以海洋模式鱼类黑点青鳉的Rh蛋白家族为研究对象，拟利用基因克隆获得其基因；定量PCR和整胚原位杂交研究其时空表达；卵母细胞表达体系和双电极电压钳研究其功能特征，实现从多层面（假基因化、亚功能化、新功能化）上研究Rh蛋白家族的功能差异，并利用生物信息学分析和突变实验（定点突变和环区替换）探讨功能分化的机制，从而系统地阐述Rh蛋白基因重复后的功能分化和分化机制。该项目的完成将加深我们对进化生物学核心问题的认识，也将阐明Rh蛋白的作用机制以及海水鱼类氨氮转运排泄的机制，进而揭示鱼类独特的氨氮代谢和高氨氮耐受性的生理适应机制。

Teleosts are unique ammoniotelic vertebrate with specific mechanism of ammonia metabolism and relatively high tolerance to high ammonia environment. It has been proved that Rhesus (Rh) proteins are membrane channels which participate in ammonia transportation and excretion in fish. Rh proteins family consist several evolutionary relative members, whose functional diversification and underlying mechanism remain unknown. In this study, we aim to uncover those questions by a coherent examination of the Rh protein family from Oryzias melastigma, an ocean model fish. The genes of Rh protein family will be cloned firstly. It follows the analysis of spatial and temporal expression of these genes via quantitative real time PCR and whole mount in situ; the elaboration of the functional characteristics via the oocyte expression system and two electrode voltage clamp technique. Multiple dimensions (pseudogenization, subfunctionalization, neofunctionalization) will, therefore, be approached in revealing the functional diversification of Rh protein family. By combining bioinformatics and mutation techniques (site-specific mutagenesis and loop substitution), the underlying mechanism will further be revealed. The achievements of this project will not only contribute to our understanding of proteins functional diversification, the central issue in evolutionary biology, but also help to elucidate the mechanism of Rh proteins and ammonia transportation and excretion in marine fish, and to reveal the physiological basis of its high tolerance to ammonia.