Abstract: 【Objective】NAC transcription factors belong to a unique class of transcription factors in plants. The common characteristics of the NAC proteins are the presence of a conserved NAC domain, comprising about 150 amino acids in N-terminals and a highly variable transcriptional regulation region in C-terminals. Extensive studies have revealed that NAC transcription factors not only play important roles in plant growth and development, but also have functions in regulation of responses to biotic and abiotic stresses. The objective of this study is to analyze the sequence feature and expression patterns of MsNAC2 gene with NAC transcription factor from alfalfa (Medicago sativa L.) and to illustrate the stress-resistance regulation mechanism of alfalfa through over-expression in tobacco, and through transgenic technology to improve the resistance of alfalfa and lay a foundation for the research on improving the quality.【Method】 The full length cDNA of MsNAC2 gene was cloned by RT-PCR and RACE, whose nucleotides and amino acid sequence were analyzed by bioinformatics software. The relative quantitative expression of MsNAC2 was detected by real-time PCR technology. A fusion expression vector MsNAC2-GFP was constructed to identify the subcellular localization. And a plant over-expression vector pBI121-MsNAC2 was constructed and used to transform tobacco by agrobacterium-mediated method. The phenotypic and physiological performance of the transformants were characterized under various abiotic stresses to investigate the function of MsNAC2 on stress resistance in tobacco. 【Result】 The full-length cDNA of MsNAC2 was 1 358 bp and the open reading frame was 1 023 bp, which encoded MsNAC2 comprising 340 amino acids with a molecular weight of 39.4 kD. The N-terminal amino acid residue of MsNAC2 contained a typical NAC conserved domain, and the C-terminal amino acid residue was highly variable. Phylogenetic analysis demonstrated that MsNAC2 shared high homology with CsNAC (Citrus sinensis Osbeck), and both belong to ATAF subfamily of NAC family. Subcellular localization analysis revealed that MsNAC2 was expressed in the nucleus. Transcription and expression analysis by Real-time PCR showed that MsNAC2 was up-regulated by 250 mmol·L^-1 NaCl, 20% PEG6000, 0.1 mmol·L^-1 ABA and 4℃, and the expression of MsNAC2 in root was apparently higher than those in leaf. The seedling height, root length, fresh weight and dry weight and other growth indicators of the transgenic tobacco were higher than that of the wild type in the treatments of NaCl, PEG6000, and 4℃. The physiological analysis revealed that malonaldehyde (MDA) was lower in the transformants than that in the WT, 82.60%, 73.20% and 77.80% in the treatments of NaCl, PEG6000, and 4℃, while the concentrations of proline, SOD and POD were increased. 【Conclusion】A new NAC transcription factor MsNAC2 was cloned from alfalfa, which responded to cold, drought and ABA. And MsNAC2-over- expressed tobacco possessed better anti-salt, anti-drought and anti-cold activities than wild-type tobacco. Those showed that the MsNAC2 might play an important role in stress response which indicated that this gene might participate in regulation of plant abiotic stress response.

Key words: alfalfa, MsNAC2, gene clone, function analysis