Objective Salt stress is an important factor affecting plant growth, and galactinol synthase (GOLS) plays an important role in plant response to abiotic stress. This study explored the role of GOLS gene in salt stress response process of alfalfa, aiming to lay a theoretical foundation for revealing molecular regulatory mechanism of salt tolerance in alfalfa and provide valuable genetic resources for stress-resistant molecular design breeding of alfalfa.

Method Alfalfa MsGOLS2 gene was cloned, gene sequence and protein structure were analyzed by bioinformatics software, an evolutionary tree was constructed, and subcellular localization was conducted. The expression specificity of MsGOLS2 in different organs of alfalfa under salt stress was analyzed by fluorescence quantitative real-time PCR. The MsGOLS2 gene of alfalfa was heterogeneously expressed in arabidopsis and subjected to salt stress treatment, the effects of overexpression of MsGOLS2 on salt tolerance of arabidopsis were analyzed at phenotypic, physiological and molecular levels.

Result (1) The total length of MsGOLS2 coding region sequence was 987 bp, encoding 328 amino acids. MsGOLS2 was a hydrophilic intracellular protein and closely related to GOLS2 in Medicago truncatula and soybean. (2) The subcellular localization results showed that MsGOLS2 was expressed in both cytoplasm and nucleus. (3) The expression level of MsGOLS2 was the highest in roots of alfalfa and significantly increased under salt stress. (4) The main root length of arabidopsis plants overexpressing MsGOLS2 was significantly higher than wild-type plants. After salt stress, the malondialdehyde content in arabidopsis overexpressing MsGOLS2 was lower than wild type, while the proline content, peroxidase and superoxide dismutase activities were higher than wild type. The changes in expression levels of antioxidant oxidase-related genes were consistent with changing trend of enzyme activities.

Conclusion Alfalfa MsGOLS2 can respond to salt stress and enhance the plant’s salt tolerance. This study can not only provide an important basis for analysis of gene function of MsGOLS2 in alfalfa, but also offer key candidate gene resources for screening, breeding, innovation and improvement of salt-tolerant germplasm resources in alfalfa.