Designing of a 35S::DREB1A molecular construct to produce environmental stress tolerant plants

Environmental stresses affect plant growth and cause losses worth hundreds of million dollars to agricultural industry each year. Many genes are induced in response to environmental stresses. The DREB1A gene is a stress-inducible transcription factor which its ectopic over-expression improves plant tolerance to environmental stresses. To produce environmental stress tolerant plants carrying the DREB1A gene, the full length cDNA of the DREB1A gene was amplified from Arabidopsis thaliana Col-0 plants by gene specific primers and cloned into pGEMT-Easy vector, and transformed into E.coli. Presence of the DREB1A gene was confirmed by restriction analysis as well as DNA sequencing. A 668-bp XbaI/BamHI digested fragment of DREB1A gene from the pGEMT::DREB1A construct was sub-cloned into the pBI121 binary vector. The recombinant plasmids were transferred into Agrobacterium tumefaciens cells (strain LBA4404) and screened on LB medium supplied with kanamycin/rifampicin (50 mg/l). Positive bacterial colonies were selected based on colony-PCR analysis and saved for further application in plant materials.