红毛菜质膜-H+-ATPase在盐胁迫响应中的作用

Bangia Lyngb. is a worldwide primitive red algal genus. The marine species was originated from about 1200 Ma ago. The freshwater species was originated from geological sequestration from marine during 174 Ma - 265 Ma ago. The freshwater species is morphologically difficult to be distinguished from the marine one. Besides, the freshwater species can adapt to seawater and the marine species can adapt to freshwater step by step. There’s no natural distribution of Bangia sp. in estuary, brackish and saline water. It indicates that Bangia sp. have evolved specific mechanisms to adapt to the completely distinct salinity. Except for their ecological specialty, the marine Bangia fuscopurpurea is a highly-valued farmed seaweed. We have studied the photosynthetic reactions and genes expression in hypo-salinity treated B. fuscopurpurea and found that expression of the plant ‘master enzyme’ plasma membrane (PM) H+-ATPase genes were up-regulated the most among all the significantly differentially expressed genes, with the mRNA abundance dozens to hundreds folds higher than the metabolic pathway genes. Aiming to investigate the function of Bangia sp. PM H+-ATPase towards salinity stress, first, the whole DNA sequences including the flanking regulation region of PM H+-ATPase genes will be cloned from both marine and freshwater Bangia species and the change of PM H+-ATPase gene transcription, protein expression and enzyme activity under salinity stress will be studied in order to reveal the evolutionary properties of this enzyme and its function towards environmental adaptation. Second, the biophysiologic and biochemical response of Bangia sp. towards distinct salinity changes and the function of PM H+-ATPase will be studied by monitoring the changes of the ROS-redox system, the energy applying system of photosynthesis and respiration, and secondary transport of ions and small substances and metabolome in hyposalinity / freshwater treated marine Bangia species and hypersalinity / seawater treated freshwater Bangia species, which was compared with the control with inhibited PM H+-ATPase activity. We hope that the study will facilitate revealing the complex signaling network of Bangia under salinity stress and help to elucidate the mechanisms of marine organisms evolving and adapting to freshwater.

红毛菜属起源于约12亿年前，中生代时期因地质隔离演化出淡水种。海水种和淡水种形态上难以区分，可适应彼此的盐度，在（半）咸水、河口鲜有分布。可见海水种演化到淡水种形成了一套特殊的适应机制。我们前期研究发现质膜H+-ATPase基因在低盐胁迫的海水种中上调表达幅度最大，比物质代谢基因高几十至上百倍。为深入探索质膜H+-ATPase在红毛菜盐胁迫响应中的作用，本项目拟：分别从红毛菜海水种和淡水种中克隆该酶基因全序列及其侧翼调控区，从基因转录、蛋白表达到酶活水平逐层解析该酶对盐胁迫处理的响应，以掌握其结构与功能的演化特征；从氧化还原应激系统、驱动酶促反应的能量供应体系、由质膜H+-ATPase调控的次级运输与物质代谢展开研究，系统论证红毛菜属对盐度巨变的响应特征以及质膜H+-ATPase的调控作用，为深入挖掘红毛菜响应盐胁迫的信号转导网络、揭示真核生物由海洋向淡水演化的适应机制奠定基础。

红毛菜属起源于约12亿年前，中生代时期因地质隔离演化出淡水种，也是红毛菜科唯一一个淡水种。红毛菜海水种和淡水种形态上难以区分，可适应彼此的盐度，在（半）咸水、河口鲜有分布。可见海水种演化到淡水种，形成了一套特殊的适应机制。研究红毛菜对盐度巨变的适应机制不仅可丰富潮间带藻类的逆境胁迫适应机制研究，并对揭示真核生物由海洋向淡水演化的适应机制具有重要意义。而国内外有关红毛菜的研究集中在系统发育上，关于两种红毛菜对盐度变化的适应机制研究少且未深入机理机制层面。本项目围绕质膜-H+-ATPase，从氧化还原应激系统、驱动酶促反应的能量供应体系、由质膜H+-ATPase调控的次级运输与物质代谢展开研究，系统论证了红毛菜属对盐度巨变的生理响应特征以及质膜H+-ATPase的调控作用，绘制了红毛菜质膜-H+-ATPase介导的盐胁迫响应网络：胞外盐度变化信号进入胞内，光合系统的光化学反应短时即受到抑制，而快速启动非光化学反应，以抵御外界胁迫；PMHA基因表达被上调；代谢组发生改变，葡萄糖-6磷酸积累，由此可促进：糖酵解和磷酸戊糖途径，前者产生大量ATP以驱动质膜H+-ATPase；后者产生大量NADPH，参与脂类代谢，不饱和脂肪酸代谢活跃（总体上调），同时作为GR的辅酶NADPH上调可促进GR活性，GSH积累；其他抗氧化酶POD/SOD/CAT的活性也随盐度变化增强，自由基被及时清除，确保了质膜和胞内膜系统上的脂质不被氧化，质膜上不饱和脂肪酸比例的维持和增加又促进质膜H+-ATPase活性，调节胞内外H+流向和流量，从而调节Na+流以及胞内Na+水平，在遭遇盐度巨变后短时间内（6h）即恢复胞内Na+平衡，光合作用也得以快速恢复。以上研究结果撰写了研究论文6篇，已发表4篇，为深入挖掘红毛菜响应盐胁迫的信号转导网络奠定了基础；并形成水产行业标准1项，将显著推动红毛菜养殖产业的发展。