Cloning And Functional Analysis Of A LbDREB Gene From Limonium Bicolor

An EST sequence encoding DRE-binding transcription factor was cloned from cDNA library, and obtained the full-length cDNA sequence by RACE method from L. bicolor, designated as LbDREB (FJ872558). The cDNA sequence of the LbDREB is 1148 bp in length, including 44 bp of the 5' untranslated region and 33 bp of the 3' untranslated region. The open reading frame (ORF) is 1071 bp in length, encoding a deduced amino acid sequence of 356 residues, with a molecular weight of 38.7 kDa and an isoelectric point of 5.51. Analysis of amino acid sequence suggested the homology of LbDREB and other DREBs from other plants was lower.We studied the expression pattern by using real-time RT-PCR, and the results suggested that LbDREB was response to NaCl, KCl, Na₂CO₃, NaHCO₃, PEG6000, low tempreture, CuSO₄ and CdCl₂. A subcellular localization on assay with GFP report gene revealed that the LbDREB-GFP fused protein was targeted to nucleus.The LbDREB gene was inserted into the yeast expressive vector pYES2 and named pYES2-LbDREB. The recombinant plasmid (pYES2-LbDREB) and pYES2 were transformed into Saccharomyces cerevisiae INVScl to characterize the tolerance of LbDREB to different abiotic stresses. The results demonstrated that LbDREB exhibits multiple abiotic stress tolerance, including salt, saline-alkali, drought, freezing, NaHCO₃, Na₂CO₃ and heavy metal.To further characterize LbDREB's function under salt and heavy metal stresses, the transgenic plants for the LbDREB gene were generated by an Agrobacterium mediated method. Six transgenic lines were selected for the test of NaCl and CuSO₄ tolerance. MDA content, SOD activity, soluble protein content, proline content and metal inos content were measured under these two stresses. The results showed that MDA content in most transgenic lines was lower than that in untransgenic tobacco under NaCl or CUSO₄ conditions. Furthmore, superoxide dismutase (SOD) activity, proline and soluble protein contents in most transformed plants were higher than that in untransgenic tobacco under salt and CUSO₄ stress. In addition, the transgenic lines retained less Na⁺ and more K⁺ after salt stress. Under CUSO₄ stress, the ratio of K⁺ to Na⁺ in all transgenic lines was higher than that in untransgenic tobacco.Based on the above results, three transgenic lines were selected for investigating the mechanism of LbDREB in regulation of downstream genes. Under the normal condition, the expression of TOBPXD (D11396),peroxidase (AB044154), peroxidas (PER4-9) (AY032675), glutathione S-transferase (GST) (D10524), V-ATPase subunit G (AJ005900) in most transgenic lines were lower than that in untransformed tobacco. After NaCl stress, the expression of late embryogenis abundant protein 5 (Lea5) (AF053076), TOBLTP (D13952), lipid transferase (ltp1) (X62395), H+-ATPase B subunit (AF220611) and H⁺-ATPase (X66737) in transgenic lines L5 and L23 were significantly higher than that in untransformed tobacco. After CuSO₄ stress, compared with untransgenic tobacco, copper/zinc superoxide dismutase (Cu/ZnSOD) (EU123521), TOBPXD (D11396), late embryogenis abundant protein 5 (Lea5) (AF053076) and TOBLTP (D13952) in all transgenic lines were up-regulated. These results suggested that over-expression of LbDREB could enhance the expression of some stress-related genes and therefore enhanced the tolerance of transgenic lines.