盐碱环境下青海湖裸鲤肠道HCO3-分泌相关基因表达差异

Differential expressions of genes related to HCO_3^- secretion in the intestine of Gymnocypris przewalskiii during saline-alkaline water transfer

以青海湖裸鲤(Gymnocypris przewalskii)为研究对象,采用半定量和定量PCR法研究HCO-3分泌相关基因SLC4(solute carrier family 4)和SLC26(solute carrier family 26)家族slc4a1、slc4a2、slc4a4和slc26a6基因组织分布情况,并对不同盐碱环境下肠道中SLC基因家族的表达情况进行定量分析,揭示青海湖裸鲤适应盐碱环境的肠道调节机制。结果表明,slc4a1、slc4a2、slc4a4和slc26a6基因在青海湖祼鲤鳃、肝脏、肾脏和肠道等多个组织中均有表达,且在肠道中的表达量较高,其中slc26a6在肠道中高表达,而在鳃中表达量极低,表现出组织特异性;在碱度组[盐度1.31±0.02,碳酸盐碱度(30.66±0.08)mmol·L-1]、盐度组[盐度15.02±0.02,碳酸盐碱度(2.12±0.05)mmol·L-1]和湖水组[盐度14.84±0.03,碳酸盐碱度(29.57±0.11)mmol·L-1]青海湖裸鲤肠slc4a1、slc4a2、slc4a4和slc26a6基因的表达量在胁迫4 d过程中均呈现出先升高后回落的现象,其中湖水组裸鲤肠SLC4、SLC26家族基因表达量上调最为明显,尤其是slc26a6基因表达量最高上升为对照组的4.9倍,同时,湖水组裸鲤直肠排泄HCO-3浓度也最高,说明盐碱环境下青海湖裸鲤通过肠道Cl–/HCO-3交换子(slc4a1、slc4a2、slc26a6)、Na+–HCO-3联合转运子(slc4a4)分泌和排泄机体内积累的碱,这一调节途径有助于青海湖裸鲤补偿因水环境中盐碱度升高而造成的渗透及酸碱失衡。

The high salinity and alkalinity of the saline-alkaline water is the primary factor restricting the survival and growth of aquatic organisms. Gymnocypris przewalskii is the most important economic fish in Qinghai Lake, a high saline-alkaline water body （pH 9.3, salinity 15 ） in China. In recent years, the population of G. przewalskii has sharply declined due to the destruction of spawning habitat, long-term overfishing and the increasing salinity and alkalinity. Thus, researches on the molecular response of G. przewalskii under saline- alkaline conditions are significant for the recovery of this endangered species. The paper is to clarify how the intestine participates in the osmotic and acid-base balance regulation of G. przewalskii upon transfer to saline and alkaline conditions by physiological and molecular methods. When freshwater-acclimated G. przewalskii were transferred to saline water[ salinity 15.02 ±0.02, carbonate alkalinity （2.12± 0.05 ） mmol· L-1 ], alkaline water [ salinity 1.31 ± 0.02, carbonate alkalinity （ 30.66 ± 0.08 ） mmol· L- 1 ] or lake water [ salinity 14.84 ± 0.03, carbonate alkalinity （29.57 ±0.11 ）mmol · L- 1 ] , there was a significant increase of base （ HCO3- ） excretion into the rectal fluids. Real-time fluorescence quantitative PCR method was used to determine the relative mRNA expressions of slc4al, slc4a2, slc4a4 and slc26a6 genes in the intestine after transfer. Among them,slc4al, slc4a2 and slc26a6 were C1^- -HCO3- exchangers, and slc4a4 was a Na ＋ -HCO3- co-transporter. Results showed that the four genes were expressed in multiple tissues, and highly expressed in the intestine, while slc26a6 expression in the gill was the lowest, exhibiting tissue specificity. The significant changes in relative mRNA expressions of investigated genes in the mid-intestine were detected on the third day of alkaline water exposure except the slc4al gene. While transferred to saline or lake water, the relative mRNA expressions of all investigated genes in the mid-intestine were significantly elevated on the second day. Among them, those exposed to lake water showed the highest level of relative mRNA expressions. The increase of SLC gene family mRNA expressions were consistent with the increase in rectal base secretion following saline-alkaline water transfer. The mRNA expressions of all investigated genes in the mid-intestine almost recovered to the original level on the fourth day, suggesting that G. przewalskii was capable of tolerating high saline-alkaline stress. These results revealed that, when freshwater-acclimated G. przewalskii were exposed to saline water, they up-regulated the expression of SLC4 and SLC26 gene families in the intestine to increase the synthesis of relevant transporters, which could aid to excrete alkaline substances （ HCO3- ） into the rectal fluid and stimulate the intestinal water absorption. In addition, we also found an increase in rectal base secretion and up-regulation of these genes after transferring to alkaline water with low salinity and high alkalinity. These indicated that SLC4 and SLC26 gene families also played important roles in acid-base regulation. Hence, in G. przewalskii, the SLC4 and SLC26 gene families were important internal factors for high tolerance of salinity and alkalinity. At the same time, the coordination of acid-base regulation, osmoregulation and ion-regulation was the basis for aquatic organisms to survive in saline-alkaline environment. Multiple tissues, genes and proteins may participate in this complex physiological process.