Overexpression SOD2 Improved Salt Tolerance In Arabidopsis Thaliana

Soil salinity is one of the most significant abiotic stresses facing crop plants in agricultural fields worldwide. High levels of sodium ion (Na+) are toxic to plants because of their adverse effects on celluar metabolism and ion homeostasis. Therefore, maintaining low levels of Na+in the cell, specifically in the cell cytoplasm, is essential for plants. Plants are thought to remove Na+ from the cytoplasm by transporting it into the vacuolar or out of the cell using Na+/H+ exchangers localized in the vacuolar and plasma membranes, respectively. SOS1 encoding a plasma membrane Na+/H+ antiporter and AtNHXl encoding a vacuolar Na+/H+ antiporter were isolated from glycophytic Arabidopsis thaliana, and overexpression of AtNHXl and SOS1 in Arabidopsis thaliana increased the salt tolerance of transgenic plants significantly. SOD2 from Schizosaccharomyces pombe, which encodes a protein with homology to bacterial and mammalia Na+/H+ antiporters in plasma membrane and responses to efflux of Na+, is an important gene involving in homeostasis. Though SOD2 gene has been cloned, ectopic expression of SOD2 in high plants has not been reported. Gene engineering based on reducing Na+ toxicity creats a new approach to plant salt tolerance. This is the first time expressing of SOD2 in Arabidopsis thaliana and maybe attribute to research the ion homeostasis in the cell.The SOD2 gene was isolated from S. pombe by RT-PCR method and confirmed by sequencing. The open reading frame of SOD2 encodes a protein about 51KD and composes of 1404bp. The SOD2 PCR product was inserted into binary plant vector pROKII. The resulting plasmid, named pROK-SOD2, was mobilized to Agrobacterium tumefaciens strain GV3101 used for plant transformation. The yeast SOD2 gene was introduced into Arabidopsis thaliana (ecotype Landsberg erecta) by Agrobaterium tumefaciens-mediated transformation with floral-dipping method under the control of CaMV 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin (30mg/L), several homozygous lines that were all tolerant to kanamycin were selected and used for further molecular andphysiological determination. Whether the gene was transformed into the Arabidopsis was confirmed by PCR-Southern blotting , Southern blotting and Northern blotting.1. PCR-Southern blotting showed that all kanamycin-tolerant plants had strong positive signals, and no signal was shown in wild type plants.2. Southern blotting analysis indicated that SOD2 gene had been integrated into all of the transgenic plants'genomes. The copy number of SOD2 in transgenic plants was mostly 1 to 5 by Southern analysis.3. Northern analysis revealed the expression of SOD2 mRNA in T3 plants of several non-segregation transgenic lines while no signal was shown in wild type plants.Several samples of transformants were self pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance.1. On MS medium containing different levels of NaCl (0-200mmol/L), overexpression of SOD2 in Arabidopsis improved seeds germination and seedling salt tolerance.2. Under different LiCl conditions (0-20mmol/L), expression of SOD2 improved lithium tolerance.3. After the treatment of different concentrations NaCl (0-200mmol/L), the fresh weight of the transgenic lines was dramaticly higher than that of wild type plants, the dry weight had no markedly differences between the transgenic plants and wild plants.4. In this work, the transgenic plants showed a tendency to accumulate less Na+ and more K+ under saline condition than wild type plants and there was a high cytosolic K+/Na+ ratio in the roots of transgenic lines.5. x-ray microanalysis of Na+, K+ contents of the symplast and apoplast showed that transgenic lines accumulate less Na+ and more K+ in the symplast than wild type plants.6. The results of the effect of NaCl concentrations on photosynmetic rate (Pn), stomatal conductance (Gs) and substomatal CO2 concentration (Ci) showed that the Pn of t...