Expression, purification and molecular characterization of a novel transcription factor KcCBF3 from Kandelia candel.

Kandelia candel, a major species of mangrove in the tropical and subtropical area, is susceptible to low temperature in winter. K. candel was introduced into Zhejiang Province (the northern margin of South China) several decades ago, and suffered from low temperature causing growth retardation, in server cases, even death. To explore the molecular mechanisms of cold acclimation in K. candel, a novel C-repeat binding factor gene KcCBF3 (Genbank accession no. KF111715) of 729 bp open reading frame (ORF) encoding a protein of 242 amino acid residues was isolated, expressed, purified and characterized. Multiple sequence alignment analysis revealed that KcCBF3 contained a highly conserved AP2/EREBP DNA-binding domain which consisting of 79 amino acid residues, as well as two CBF signature sequences. Phylogenetic analysis indicated that KcCBF3 belonged to the A-1 subgroup of DREB subfamily based on the classification of AP2/EREBP transcription factors in Arabidopsis. Semi-quantitative RT-PCR showed that KcCBF3 transcripts were highly accumulated in roots and leaves, and could be induced by low temperature. Electrophoresis mobility shift assay (EMSA) demonstrated KcCBF3 could bind to the core sequence (CCGAC) of cis-acting element C-repeat (CRT)/dehydration-responsive element (DRE) in vitro. These results implied that KcCBF3 might participate in the adaptation of K. candel to low-temperature stress by binding to CRT/DRE element.