Abstract：As one of the most important transcription factor families in plants, MYB plays a crucial role in regulating plant growth and development as well as responding to abiotic stress. In this study, the MYB gene family in S. australis was systematically investigated through genome-wide identification and characterized by bioinformatics analyses, which led to the identification of 104 SaMYB genes classified into 4 subfamilies: 1R-MYB (34 members), R2R3-MYB (66 members), 3R-MYB (3 members), and 5R-MYB (1 member). Phylogenetic analysis divided the R2R3-MYB members into 20 distinct subgroups; members within each subgroup shared comparable gene structures and conserved motifs. Physicochemical profiling demonstrated that SaMYB proteins exhibited isoelectric points (pI) ranging from 4.69 to 9.7, amino acid lengths varying between 89~1 563 aa, and molecular weights spanning 9.99~170.02 kD. Synteny analysis identified 96, 89, and 47 homologous gene pairs between S. australis and B. vulgaris, A. thaliana, and O. sativa, respectively. The SaMYB gene family underwent 10 segmental and 3 tandem duplication events, with 8 gene pairs under purifying selection. Cis-element analysis demonstrated that SaMYB promoters were enriched with regulatory elements responsive to phytohormones and abiotic stresses. Transcriptome sequencing and qPCR validation revealed significant upregulation of Sau00119, Sau00120, Sau19637, Sau02923, Sau08091 and Sau13875 under salt stress conditions. Agrobacterium-mediated overexpression of Sau19637 in Nicotiana benthamiana significantly activated the expression of the downstream CBF4 gene.Yeast one-hybrid assays confirmed that Sau19637 could directly bind to the CBF4 promoter to regulate its transcription, thereby enhancing plant salt tolerance. This study provides new candidate genes and regulatory targets for further elucidating the molecular mechanisms of salt tolerance in S. australis.
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