Cloning And Functional Characterization Of An LbGST Gene From Limonium Bicolor

Plant glutathione S-transferases (GSTs) play an important role in plant stress responses and growth development. In the present study, a novel GST gene, designated LbGST (GenBank number FJ620899), was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). The full-length cDNA sequence of LbGST is 987 bp in length, including 29 bp of the 5' untranslated region and 307 bp of the 3'untranslated region. The open reading frame (ORF) is 651 bp in length, encoding a deduced amino acid sequence of 216 residues with a molecular weight of 24.5 kDa and an isoelectric point of 5.98. Mutiple sequenc alignment analysis suggested that the homology between LbGST and other GSTs from other plants was low.the expression pattern of LbGST in response to abiotic stress was researched by using real-time RT-PCR. The results suggested that LbGST can response to the treatments of NaCl, NaHCO3, PEG6000 and low temperature. Subcellular localization assay revealed that the accumulation of LbGST protein was found in the nucleus.The LbGST gene was inserted into the prokaryotic expressive vector pGEX-4T-2, and transformed into E. coli BL21. The expression of LbGST gene in E. coli BL21 was induced by 0.01mmol/L IPTG. SDS-PAGE results indicated that a specific band, with molecular weight of 50 kDa, displayed by transformants was the target peptide.The LbGST gene was inserted into the yeast expressive vector pYES2, and the construct was named pYES2-LbGST. The recombinant plasmid (pYES2-LbGST) and pYES2 were transformed into Saccharomyces cerevisiae INVSc1 to characterize the tolerance of LbGST to different abiotic stresses. The results demonstrated that LbGST gene exhibited multiple abiotic stress tolerance, including salt, drought, freezing, NaHCO₃, heat and ultraviolet radiation.To further characterize its function under salt, the LbGST gene was inserted into the plant expressive vector pROKII for plant transformation. The LbGST transformed plants were generated by an Agrobacterium mediated method. The transgenic lines were examined by PCR and Northern hybridization, confirming that LbGST gene has been integrated into the genome of tobacco and successfully expressed in all the transgenic lines.In addition, five transgenic lines were selected for NaCl tolerance test. GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), proline and metal inos were measured and analysised under salt stress. The results showed that GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity in transformed plants were significantly higher than in wild type (WT) plants, especially under salt stress. However, malondialdehyde (MDA) content in transgenic plants was lower than that in WT plants under NaCl condition. Furthermore, the Na+content in transgenic plants was much lower than that in WT plants under stress condition. The results suggested that overexpression of LbGST gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST gene mediates many physiological pathways to enhance stress resistance in plants.