Isolation and characterization of two Medicago falcate AP2/EREBP family transcription factor cDNA, MfDREB1 and MfDREB1s

DREB transcription factors play an important role in tolerance to abiotic stress in high plants. In this work, two new DRE-binding protein genes MfDREB1 and MfDREB1s cDNA that encoded an AP2/EREBP type transcription factor were isolated by RT-PCR from Medicago falcate seedlings. Sequence analysis showed MfDREB1 and MfDREB1s were almost identical except that there was a 202 bp fragment at the 3′ end of the MfDREB1s cDNA that is absent in MfDREB1 cDNA. The MfDREB1 has a open reading frame of 651 bp, which encodes 216 amino acid residues. The putative protein is deduced a predicted molecular mass of 24.6 kDa and a pI of 5.95. The MfDREB1s has a open reading frame of 555 bp, the putative protein is 184 amino acid long with a predicted molecular weight of 20.8 kDa, pI 9.11. The Protein Blast data revealed that the two proteins can be classified as a typical member of the AP2/EREBP family of DNA-binding proteins. The comparison of the MfDREB1 cDNA and MfDREB1s cDNA with their corresponding genes in genomic DNA showed that the size and nucleotide sequence of the cDNA was identical to that of the genomic DNA. This suggested that the genomic MfDREB1 gene and MfDREB1s gene had no introns. Southern blot analysis indicated that MfDREB1 and MfDREB1s are multi-copy genes in Medicago falcate genome. Northern blot analysis indicated that the MfDREB1 and MfDREB1s genes were induced by low temperature stress.

Research highlights
► In this manuscript, we isolate two MfDREB genes from M. falcate named MfDREB1 and MfDREB1s. All of them were induced by cold and salt stress but not by drought.
► Sequence analysis shows MfDREB1 and MfDREB1s are almost identical except that there is a 202 bp fragment absences at the 3′ end of the MfDREB1s cDNA in contrast with the MfDREB1 cDNA. The MfDREB1 cDNA have 92.88% of its identity in common with the MfDREB1s cDNA. Southern blot analysis indicated that MfDREB1 and MfDREB1s are multi-copy genes in Medicago falcate genome. PCR with genomic DNA as a template shows MfDREB1 and MfDREB1s are two independent genes in M. falcate genome, and RT-PCR analysis shows that each of them has its own transcripts. The particular relationship of the two MfDREB genes has not been reported in other plants.