Isolation and molecular characterization of wheat (Triticum aestivum) Dehydration Responsive Element Binding Factor (DREB) isoforms

Due to adverse effects of abiotic stresses on plant growth and development, we focused on an effective abiotic stress responsive element known as Dehydration responsive element binding factor (DREB) in Triticum aestivum. The objective of the research was to isolate and molecularly characterize two wheat DREB2 (WDREB2) gene isoforms. In order to isolate WDREB2 isoforms, plants were exposed to cold stress. Total RNA was extracted from cold-treated plant leaves and first-strand cDNA was synthesized. Two isoforms were isolated by specific primers and submitted in NCBI database (Accession No. JQ004969 and HQ171443). Searching for similar sequences to isolated isoforms by NCBI BLASTn resulted in 10 sequences in Triticum aestivum with ≥ 95% identity and ≥ 90% coverage. Multiple alignments on homologous sequences were preformed. The results showed that all these homologous sequences are related to one definite gene, WDREB2, which has 3 different alternate spliced forms or isoforms. β Isoform that lacks transcription activation domain is inactive while α is an active isoform. In order to verify α isoform activity, WDREB2 α isoform was expressed in tomato using Agrobacterium mediated transformation and α isoform-expressed plants were tested. Phonotypical comparison between transgenic and normal plants under cold stress confirmed the activity of WDREB2α isoform. In addition to laboratory research, 2 available sequences, submitted as wheat DREB gene promoter (Accession No GU785009 and GU785008) were selected and promoter analysis was carried out. Several motif sites such as ABRE, C-repeat/DRE and MBS, which are important Cis acting elements in abiotic stress response were identified.