高碳酸盐碱胁迫对尼罗罗非鱼氨代谢基因表达变化的影响

Change in ammonia metabolism gene expression of Oreochromis niloticus under the stress of high carbonate alkalinity

为了解尼罗罗非鱼（Oreochromis niloticus）在碱环境适应过程中氨代谢调节途径,本研究选取了5个氨代谢相关酶：谷氨酰胺合成酶（glutamine synthetase,GS）、碳酸酐酶5A（carbonic anhydrase 5A,CA-5A）、谷氨酰胺酶（glutaminase 2,GLS2）、氨甲酰磷酸合成酶（carbamyl phosphate synthetase 1,CPS1）、氨转运蛋白（ammonium transporter Rh type C-2 like,Rhcgl2）,研究了急性碳酸盐碱度胁迫条件下,尼罗罗非鱼血氨浓度变化、氨代谢相关酶基因表达水平及其酶活性变化。结果表明,随碳酸盐碱胁迫浓度升高,尼罗罗非鱼血氨浓度上升,随时间推移呈先上升后下降的变化趋势,在胁迫后12 h达到峰值。氨代谢相关基因在不同碱度下、不同组织中均有不同程度的上调表达,随着胁迫时间推移呈先上升后下降的变化趋势,胁迫后12-24 h各基因表达水平显著升高,随后逐渐恢复到稳定水平;氨代谢相关基因具有一定的组织表达差异：氨转运蛋白基因（Rhcgl2）主要在鳃中表达,碳酸酐酶5A基因（CA-5A）、谷氨酰胺合成酶基因（GS）、氨甲酰磷酸合成酶基因（CPS1）主要在肝中表达,谷氨酰胺酶基因（GLS2）主要在肾和鳃中表达。碳酸酐酶和谷氨酰胺合成酶活性随胁迫碱度的升高而上升,碳酸酐酶、谷氨酰胺合成酶活性变化分别在鳃、肝中最为显著。研究结果表明,碳酸盐碱度胁迫会引起尼罗罗非鱼血氨水平升高,随着时间推移血氨水平下降,推测鳃、肝、肾中不同氨代谢基因共同参与调节氨代谢,在鳃中通过直接排氨,在肝中通过合成谷氨酰胺、尿素途径,共同调节降低血氨水平。

We investigated the ammonia metabolism pathway of Oreochromis niloticu after acute carbonate alkalinity gradient stresses, and studied several ammonia metabolism-related genes： glutamine synthetase （GS）, carbonic anhydrase 5A（CA-SA）, glutaminase 2（GLS2）, carbamyl phosphate synthetase I（CPS1） and ammonium transporter Rh type C-2 like （Rhcgl2）; serum ammonia concentration; gene expression; and enzyme activity. The serum ammonia concentration increased as the carbonate alkalinity gradient rose, and showed a trend to increase and then decrease, with the greatest at 12 h. Ammonia metabolism-related genes in different tissues were up regulated to various degrees after different alkalinity stresses. The expression change trends showed an increase and then a decrease over time; their expression increased significantly at 12-24 h, then recovered gradually to stable levels. Rhcgl2 was mainly expressed in the gill; CA-SA, GS and CPS1 were mainly expressed in the liver; and GLS2 was mainly expressed in the kidney and gill. Enzyme activity results showed that CA and GS activities increased along with increased alkalinity, and that CA acted most significantly in the gill and GS in the liver. Over-all, the results showed that carbonate alkalinity stress can result in the raising of serum ammonia concentrations, and that ammonia metabolism-related genes in the gill, liver and kidney played different roles in adjusting to ammonia metabolism： excreting the ammonia directly in the gill, producing glutamine and urea in the liver, and decreasing the serum ammonia concentration.