Transcription Factor DREB1C Improves Salt Tolerance In Arabidopsis

EREBP/AP2-type proteins are members of a large DNA binding protein(DBP) family only found in plants. Some members such as AtERF, Pti, APETALA2 and AtDREB/CBF have very important regulatory functions in ethylene response, defense response, plant development control and environmental stress response, respectively.Recently, two distinct genes, DREB1 and DREB2, encoding DNA binding proteins that specifically bind to the DRE sequence of rd29A gene had been cloned from Arabidopsis by using the one-hybrid screening technique. They are involved in the activation of the rd29A gene in response to dehydration, high-salt, and low-temperature stress. The two transcription factors have no significant amino acid sequence identity. However, both the DREB1A and DREB2A proteins contain a typical EREBP/AP2 binding motif. They all belong to EREBP/AP2-type protein family. DREB1 has three homologs, named DREB1A, DREB1B and DREB1C respectively. DREB1A has been deeply studied by Yamaguchi-shinozaki and Shinozaki group in Japan. The DREB1B is identical to CBF1 which Stockinger group has also studied deeply. But there is no report on study of stress tolerance of DREB1C, which contains a single open reading frame of 216 amino acids and encodes a putative protein with a predicted molecular mass of 24.3kD.To broaden our knowledge about regulatory molecules involved in stress response, we cloned the DREB1C from Arabidopsis, and characterized its salt tolerance in transgenic research first. The flowing results were obtained:1. .The DREB1C full length was cloned from Arabidopsis genome by PCR, and was inserted into pGEM-T-easy vector. By blue-white screening and sequence analysis, we obtained the positive clone.2. We constructed plant binary expression vector carrying fusions of the enhanced CaMV 35S promoter and DREB1C (35S: DREB1C) in the sense orientation. PBI121 was chosen as binary vector. By digesting with BamHI and Sacl, we confirmed that the construction of the binary expression vector was correct.3. Arabidopsis plants were transformed with the constructed plant binary expression vector. Eighteen independent antibiotic-resistant Arabidopsis transformants for DREB1C were obtained by using a vacuum infiltration method. Transgenic plants of T3 generation were used for further analysis.4. We confirmed that the cloned gene DREB1C was really transformed into Arabidopsis by PCR method, in which sequences of CaMV 35S were used as forward primer.5. Expression of DREB1C in transgenic plants was confirmed by Northern blotting. The level of the DREB1C transcripts was higher in transgenic plants than in the wild-type plants under unstressed control condition.6. We tested the DREB1C's effect on salt tolerance. The result showed that overexpression of DREB1C could improve salt tolerance in Arabidopsis. The transgenic plants could develop well in 100 mM NaCl and 150 mM NaCl whereas control plants grew slowly and was seriously retarded; in 0 mM NaCl, transgenic plant roots are stronger than wild type..