Qiao Huiru, Zhang Depeng, Zhang Zizhao, Meng Jing, He Pin, Zhang Shichao, Wang Yan, Cai Hua, Li Yong
College of Life Science, Northeast Agricultural University, Harbin 150030, China.
Int J Mol Sci. 2025 Apr 27;26(9):4165. doi: 10.3390/ijms26094165.
The Rare Cold-Inducible 2 () gene encodes a conserved hydrophobic peptide that plays a crucial role in ion homeostasis, membrane stability, and responses to abiotic stress. In this study, six members of the gene family were identified in L., all of which contain highly conserved PMP domains. Comparative collinearity analysis revealed syntenic relationships between and , with each gene displaying distinct expression profiles under various stress conditions. Among them, was significantly upregulated in response to salt stress. Alfalfa plants overexpressing exhibited enhanced salt tolerance, as evidenced by increased antioxidant enzyme activities and reduced accumulation of malondialdehyde (MDA), hydrogen peroxide (HO), and superoxide anion (O) compared to wild-type plants. Furthermore, the transgenic lines maintained better Na⁺/K⁺ homeostasis under salt stress, reflected by a lower Na⁺/K⁺ ratio and significantly elevated expression of key ion transport genes, including , , and . To elucidate the molecular mechanisms underlying function, a yeast two-hybrid (Y2H) screen identified 151 potential interacting proteins. Gene Ontology (GO) enrichment analysis revealed that these interactors are mainly involved in antioxidant defense and ion transport. Further validation confirmed direct interactions between and both calmodulin (CaM) and vacuola H⁺-ATPase (V-H⁺-ATPase), suggesting that contributes to ion homeostasis through interactions with CaM and V-H⁺-ATPase, thereby promoting Na⁺/K⁺ balance and enhancing salt tolerance. These findings provide new insights into the role of in salt stress responses and underscore its potential as a genetic target for enhancing salinity tolerance in forage crops.
罕见的冷诱导2（）基因编码一种保守的疏水肽，该肽在离子稳态、膜稳定性和对非生物胁迫的响应中起关键作用。在本研究中，在紫花苜蓿中鉴定出该基因家族的六个成员，所有成员均含有高度保守的PMP结构域。比较共线性分析揭示了紫花苜蓿和其他物种之间的同线关系，每个基因在各种胁迫条件下表现出不同的表达谱。其中，该基因在盐胁迫下显著上调。与野生型植物相比，过表达该基因的苜蓿植株表现出增强的耐盐性，这通过抗氧化酶活性增加以及丙二醛（MDA）、过氧化氢（HO）和超氧阴离子（O）积累减少得以证明。此外，转基因株系在盐胁迫下维持了更好的Na⁺/K⁺稳态，表现为较低的Na⁺/K⁺比值以及关键离子转运基因（包括、和）的显著上调表达。为了阐明该基因功能的分子机制，酵母双杂交（Y2H）筛选鉴定出151个潜在的相互作用蛋白。基因本体论（GO）富集分析表明，这些相互作用蛋白主要参与抗氧化防御和离子转运。进一步验证证实了该基因与钙调蛋白（CaM）和液泡H⁺-ATP酶（V-H⁺-ATPase）之间的直接相互作用，表明该基因通过与CaM和V-H⁺-ATPase相互作用促进离子稳态，从而促进Na⁺/K⁺平衡并增强耐盐性。这些发现为该基因在盐胁迫响应中的作用提供了新的见解，并强调了其作为提高饲料作物耐盐性的遗传靶点的潜力。
