Abstract:

Salt stress is one of the main abiotic stress factors affecting plant growth and development. Members of the BBX family of transcription factors are known to play important regulatory roles in pigment accumulation， photomorphogenesis， seed development， and stress adaptation. To clarify the function of Medicago sativa （alfalfa） BBX genes， we used Primer Premier 5 to design specific primers according to the sequence of MsBBX24 in the NCBI database， and cloned MsBBX24 using the cDNA of alfalfa as the template. Bioinformatics software was used to analyze the sequence and structure of MsBBX24， and to compare MsBBX24 with related BBX24 sequences in other plants. A phylogenetic tree was constructed to analyze the evolutionary relationships among BBX24s. The transcriptional profile of MsBBX24 was analyzed by real-time quantitative polymerase chain reaction （qRT-PCR）. An MsBBX24-overexpression vector was constructed by DNA digestion and ligation， and introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation. The positive transgenic Arabidopsis plants were screened using the herbicide Basta and the presence of the transgene was confirmed by semi-quantitative RT-PCR. The salt tolerance of wild-type and transgenic Arabidopsis plants overexpressing MsBBX24 was analyzed on the basis of phenotypic characteristics and physiological indexes. The MsBBX24 coding sequence was 723 bp long， encoding a polypeptide of 240 amino acids with a predicted molecular weight of 30.58 kDa and theoretical isoelectric point of 7.74. MsBBX24 showed high homology with BBX24 of Cicer arietinum and Medicago truncatula. The results of qRT-PCR analyses showed that the transcript levels of MsBBX24 increased in response to salt stress （150 mmol·L^-1 NaCl）. The transgenic Arabidopsis plants showed increased tolerance to NaCl stress. Compared with the controls， the transgenic plants overexpressing MsBBX24 showed enhanced growth of seedling roots and lateral roots， a higher cotyledon greening rate， significantly reduced ion leakage， malondialdehyde content， and H₂O₂ content， increased contents of chlorophyll and proline， and higher peroxidase and superoxide dismutase activities. These results show that， under salt stress， MsBBX24 improves the antioxidant defense system to remove reactive oxygen species， thereby enhancing the salt tolerance of transgenic plants. Thus， MsBBX24 is an important candidate gene for breeding new salt-tolerant varieties of alfalfa.

Key words: alfalfa, MsBBX24, gene cloning, salt tolerance