Identification, evolutionary analysis and functional analysis of the SLC12 gene family in Gymnocypris przewalskii for saline-alkali adaptation

The solute carrier family 12 (SLC12) represents a crucial group of plasma membrane-bound transporter proteins responsible for the coordinated movement of Na⁺, K⁺, and Cl⁻ions. These evolutionarily conserved proteins play fundamental roles in maintaining cellular homeostasis, including cell volume regulation, blood pressure control, and ion balance maintenance. Gymnocypris przewalskii is capable of surviving in both freshwater and brackish water, and exhibits robust osmotic pressure adaptation and regulatory mechanisms. It is primarily distributed in Qinghai Lake and its surrounding tributaries, exhibiting an ecological habit of migrating upstream from Qinghai Lake to freshwater rivers during the spawning period, and subsequently migrating back to Qinghai Lake after completing spawning. Recent studies have revealed a significant enrichment of osmoregulation-related genes in the kidney of G. przewalskii during saline-alkali adaptation, including sodium/potassium-transporting ATPase, chloride channel proteins, and solute carrier family (SLC) genes, with the SLC family playing a pivotal role. However, the diversity and functional differentiation of the SLC superfamily in G. przewalskii remain poorly understood. Furthermore, there is limited reporting on the mechanisms by which its various subfamilies contribute to osmoregulation. The gill and kidney serve as the primary osmoregulatory organs in teleost fish. In response to fluctuations in water salinity and alkalinity, these organs perform critical functions such as nitrogenous waste excretion, acid-base balance, and ion reabsorption. Members of the SLC12 transporter family are likely instrumental in these processes. NKCC2 (SLC12A1) is expressed on the basolateral membrane of epithelial cells in the thick ascending limb of the loop of Henle, where it facilitates salt ion reabsorption. In Mus musculus, knockout of SLC12A1 severely compromises renal function, leading to symptoms such as dehydration and hypokalemia. In contrast, NCC (SLC12A3) is primarily localized to the apical plasma membrane of the distal convoluted tubule. Impairment of its function reduces the reabsorption of sodium and water in renal tissues, consequently resulting in decreased cell volume. In summary, the available evidence collectively suggests that the SLC12 transporter family plays a pivotal role in regulating cell volume and osmotic pressure in fish. This investigation sought to investigate the evolutionary mechanisms and functional contributions of the SLC12 gene family in the adaptation of G. przewalskii to saline-alkaline environments. Employing an integrated bioinformatics approach, we systematically identified and characterized SLC12 family members across three teleost species: G. przewalskii, Cyprinus carpio, and Oncorhynchus mykiss. Comprehensive phylogenetic reconstruction and structural analyses were performed, followed by tissue-specific expression profiling of 11 distinct tissues in G. przewalskii using quantitative real-time PCR (qRT-PCR), with particular emphasis on gill and kidney responses to salt and saline-alkaline stress conditions. The study identified a total of 51 complete SLC12 genes in G. przewalskii, divided into ten subtypes (SLC12A1, SLC12A2, SLC12A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC12A8, SLC12A9, SLC12A10) , consistent with those subtypes in the C. carpio. However, the O. mykiss genome lacks the SLC12A10 subfamily. Structural domain analysis revealed the characteristic presence of amino acid permease or potassium-chloride cotransporter domains across family members. Tissue-specific expression profiling demonstrated elevated expression of SLC12A2, SLC12A7, and SLC12A10 in gill and kidney tissues, while SLC12A1, SLC12A3, and SLC12A9 showed kidney-enriched expression, SLC12A5 and SLC12A6 exhibited higher expression levels in brain tissue. Differential expression patterns of SLC12 genes were observed under osmotic stress conditions. Renal tissue exhibited significant upregulation of GpSLC12A2, GpSLC12A5, GpSLC12A6, and GpSLC12A7 (P<0.05), while GpSLC12A1, GpSLC12A3, GpSLC12A4, and GpSLC12A10 demonstrated marked downregulation in response to both salt and saline-alkaline stress. However, the expression levels of GpSLC12A1, GpSLC12A2, and GpSLC12A3 showed a significant increasing trend in the gill, and the expression levels of GpSLC12A5 and GpSLC12A10 were downregulated. This study demonstrates that the SLC12 family exhibits significant differences in expression levels across different tissues of G. przewalskii and plays differential osmoregulatory roles during adaptation to saline-alkaline environments. As the first comprehensive study to characterize both sequence and expression patterns of the SLC12 gene family in G. przewalskii, this work identifies potential molecular markers for extremophile adaptation research and conservation of saline-alkaline aquatic ecosystems.