Abstract:

The NAC （NAM， ATAF1/2， CUC1/2） transcription factors are plant-specific regulators that play critical roles in plant growth and development， hormone signaling， and stress responses. Alfalfa （Medicago sativa）， one of the most important leguminous forage crops worldwide， is renowned for its high nutritional value and quality. To investigate the function of the MsNAC053 gene in alfalfa， specific primers were designed using SnapGene and NCBI to clone MsNAC053 through polymerase chain reaction （PCR） technology. The physicochemical properties， secondary structure， and subcellular localization of the protein were analyzed through bioinformatics tools. And the tissue-specific expression patterns of MsNAC053 and its responses to abiotic stresses were analyzed through quantitative real-time PCR （qRT-PCR）. The subcellular localization analysis were conducted by introducing vector into tobacco （Nicotianatabacum） leaves via Agrobacterium-mediated transformation. The results showed that the coding region of MsNAC053 was 903 bp， encoding 300 amino acids， with a predicted molecular weight of 34.62 kDa， an aliphatic index of 73.82， a theoretical isoelectric point （pI） of 7.05， a grand average hydropathicity of -0.604， and an instability coefficient of 47.28， categorizing it as an unstable hydrophilic protein without transmembrane regions. Cis-acting elements responsive to low-temperature （LTR） and abscisic acid responsiveness （ABRE） were identified. Subcellular localization confirmed that MsNAC053 was localized in nucleus. Phylogenetic and amino acid sequence analyses revealed close evolutionary relationships between MsNAC053 and other NAC proteins from leguminous species such as Medicago truncatula and Vicia villosa. qRT-PCR demonstrated tissue-specific expression of MsNAC053， with the highest expression level in young leaves and the lowest expression level in cotyledons. Drought， salt， and ABA treatments significantly upregulated the expression level of MsNAC053， indicating that it plays a role in response to drought and salt stresses. In summary， the results of this study provides both theoretical insights and a candidate gene for molecular breeding of stress-resistant alfalfa varieties.

Key words: alfalfa, NAC transcription factor, gene cloning, expression analysis, abiotic stress