高碱胁迫下达里湖瓦氏雅罗鱼Rh蛋白介导的转氨机制

Ammonia excretion is the key physiological process for fish removal of nitrogenous waste in vivo. The gill is the major site for ammonia excretion. Under high ammonia or acid water conditions, non-ionic NH3 cannot be excreted via diffusion, NH3 channels in cell membranes provides an efficient and regulated pathway. Rh proteins mediated NH3 across the gill epithelium which will be trapped via acid secretion by H+-ATPase and /or Na+/H+ exchanger (NHE) immediately. This “acid trapping” or “proton-facilitated” mechanism maintains a favorable NH3 diffusive gradient (PNH3) across the gill epithelium. However, in highly saline and alkaline lakes, this “acid trapping” will be impeded due to extreme alkalinity and high pH. Despite, most of fish with saline and alkaline tolerance actively excretes ammonia against PNH3, their mechanisms are unknown. Leuciscus waleckii from Lake Dali is of the characteristics of extreme alkalinity tolerance. The study found that Rh proteins involved in the ammonia excretion process, Na+ transporting proteins (NHE and Na+/K+-ATPase) play important roles in maintaining the Na+ net balance during the high alkali-adaptation. Therefore, we believe that the Rh protein may work with Na+ transporting proteins together to excrete ammonia. This project intends to verify if Rh proteins mediated ammonia excretion mechanism is dependent on the Na+ transporters in L. waleckii under alkali stress using the methods of the quantitative expression and cellular localization of the target gene and protein, and ultimately to clarify the ammonia mechanisms of fish with high alkali tolerance.

排氨是鱼类清除体内含氮废物的关键生理过程。鳃组织是鱼类排氨的主要部位。在高氨或强酸等逆境中，NH3无法自由扩散排出，需要Rh蛋白介导其跨膜转运，使之被H+-ATPase或Na+/H+交换蛋白(NHE)形成的H+捕获生成NH4+，并在鳃上皮细胞内形成扩散梯度 (PNH3)，从而完成排氨过程。但在高盐碱湖泊中，水体高碱度高pH会阻碍这一过程，然而大部分耐盐碱鱼类仍以抗PNH3梯度的方式主动排氨，其机制不详。达里湖瓦氏雅罗鱼(Leuciscus waleckii)极耐高碱，研究发现Rh蛋白参与其耐高碱的排氨过程，Na＋转运蛋白(NHE、Na+/K+-ATPase)在维持其体内Na+净平衡过程中发挥重要作用。据此，我们认为Rh蛋白可能和其协同作用实现排氨。本项目拟从基因、蛋白表达及细胞定位验证高碱胁迫下达里湖瓦氏雅罗鱼Rh蛋白是否与Na＋转运蛋白协同作用共同排氨，从而阐释耐高碱鱼的排氨机制。

瓦氏雅罗鱼(Leuciscus waleckii)属于鲤科鱼类，具有极强的耐高碱特性，能够耐受内蒙古达里湖碳酸盐碱度54 mM(pH9.6)的极端恶劣环境，是开展鱼类耐碱生理和分子机制研究良好的模式生物。本项目以瓦氏雅罗鱼碱水种(达里湖种群)和淡水种(松花江种群)为研究材料，以接近达里湖碱度（~50mM）为胁迫浓度，利用比较生理学方法，系统分析了两种实验鱼的组织微观结构、氨氮排泄速率、血液离子和渗透压、排氨相关基因表达等方面的生理和分子表现。研究发现，雅罗鱼碱水种在高碱胁迫120h后能够从生理和分子水平全面启动抗碱特性，保持体液稳态，实现对盐碱环境的适应，而淡水种可能需要更长的时间适应或直接面临死亡。具体来讲，高碱胁迫下碱水种鳃、肾组织都发生了适应性改变，鳃结构完整生理功能正常，肾结构萎缩抑制部分生理功能；高碱胁迫下碱水种能够提高皮质醇含量，促进离子细胞增殖，提高鳃Na+/K+-ATPase活性及基因表达，加快离子细胞排Na+保H+，排HCO3-保Cl-，提高离子和渗透压调节能力，保持体液稳态；高碱胁迫下雅罗鱼碱水种能够启动排氨，加快鳃、肾组织的排氨速率降低体内毒氨的积累；参与鳃、肾排氨的Rh基因家族(rhcg1、rhbg和rhcg2)显著高表达，NHE基因家族(nhe3a、nhe3b和nhe2, 排酸)抑制表达，SLC26a基因家族(slc26a1、slc26a5和slc26a6，排碱)显著高表达。结合氨氮排泄速率、基因表达及血液离子含量综合分析，初步判断雅罗鱼碱水种的排氨机制与海水和淡水鱼类的“H+辅助排氨机制”明显不同，推测Rh蛋白可能与鳃离子细胞的Cl-/HCO3-转运蛋白或酶类存在协同作用，共同促进排氨维持体内离子稳态。这些研究结果，为从不同层面解析雅罗鱼耐高碱过程中氨氮代谢的生理和分子机制研究奠定了基础，并提出了新见解。