Abstract:

The BBX family of transcription factors is involved in plant growth and development processes such as photomorphogenesis， flowering physiology， shade avoidance， seed development， hormone signaling， and responses to environmental stress. Medicago sativa， commonly known as alfalfa， is a forage crop with strong salt tolerance and high forage quality， and has been called the “queen of forages”. Uncovering and investigating the molecular mechanisms of the alfalfa MsBBX20 gene in response to abiotic stress can reveal the biological basis for alfalfa stress resistance and provide new genetic resources for molecular breeding for stress resistance. The MsBBX20 gene cDNA sequence was cloned using RT-PCR and 3′/5′ RACE PCR techniques， and bioinformatics analysis showed that its CDS is 834 bp long， encoding 278 amino acids， and the protein is a member of the zinc finger protein family. The expression pattern of MsBBX20 in different alfalfa tissues and under various abiotic stresses was analyzed using qRT-PCR technique. Subcellular localization was performed by transiently expressing MsBBX20 in Allium cepa epidermis via a gene gun. The MsBBX20 promoter sequence， with a length of 1737 base pairs， was cloned and its cis-acting elements were analyzed. This promoter can efficiently drive the expression of the GUS reporter gene in the leaves， stems， and roots of Nicotiana tabacum. The MsBBX20 promoter sequence was linked to the pCAMBIA-1301 vector containing the GUS gene， and then transiently transformed into N. tabacum and GUS activity in different tissues was analyzed by histochemical staining. A pCAMBIA3301-MsBBX20 plant overexpression vector was constructed and genetically transformed into wild-type Arabidopsis thaliana using the Agrobacterium-mediated method， A. thaliana lines overexpressing MsBBX20 were thus obtained. Transgenic lines of A. thaliana were treated with different concentrations of salt solution （150， 200， and 300 mmol·L^-1 NaCl） and the malondialdehyde （MDA） content and antioxidant enzyme activity of different lines were analyzed. Evolutionary analysis indicated that the MsBBX20 protein of alfalfa is most closely related to the TpBBX20 of red clover （Trifolium pratense）. The MsBBX20 protein is localized to the nucleus. The MsBBX20 gene is most highly expressed in flowers and can respond to various abiotic stresses such as drought， salt， cold， abscisic acid， gibberellin， and light. Transgenic A. thaliana lines overexpressing MsBBX20 were obtained， and three highly expressed positive lines were selected for functional validation. Under different concentrations of NaCl， the MDA content of A. thaliana lines overexpressing MsBBX20 was significantly lower than the control， while the activities of superoxide dismutase， catalase， and peroxidase were significantly higher than the control. The MsBBX20 gene， a zinc finger protein transcription factor from alfalfa， is highly expressed in flowers and can respond to a variety of abiotic stresses and exogenous hormone treatments， indicating that MsBBX20 may be involved in multiple stress response processes in alfalfa. Further research demonstrated that MsBBX20 may enhance the resistance of transgenic A. thaliana to salt stress by regulating the plant’s antioxidant enzyme system， mitigating oxidative damage to plant cells caused by stress， and thereby improving plant salt tolerance.

Key words: Medicago sativa, MsBBX20, gene cloning, salt tolerance, function analysis