Functional Characterization Of Wheat Na~+ /H~+ Antiporter TaNHX2 And Study Of TaNHX2 C- Terminus Domain

Soil salinity is one of the major factors limiting agricultural production, and sodium is one of the major components of saline soils. The area of saline soil is growing at a high speed because of the secondary salinization due to unreasonable irrigation. According to statistics, drought and salinization caused by more than 40% reduction in the impact of crop yields in the various environment factors. Consequently, how to improve salt tolerance of plants has become an increasingly imminent problem to be resolved. Genetic analysis has shown that maintenance of a low concentration of cytoplasm sodium is the key to sodium tolerance. Na~+ can enter into the cytosol by non-selective cation channels under salt stress. To reduce the harmless of sodium ions, it mainly has two methods to decrease the concentration of cytoplasm for plants. The one is that plants cells transport Na~+ from cytoplasm to the external medium by the operation of plasma membrane Na~+/H~+ antiporter. And the other is that plants cells compartmentalize it into vacuole by the tonoplast-bound Na~+/H~+ antiporter, thus maintaining the cytosolic ion homeostasis. As a non- halophyte, wheat production is affected by salt stress and its growth is severely inhibited under salinity condition. Here we cloned the Na~+/H~+ antiporter gene TaNHX2, the function of TaNHX2 and its C-terminus domain were studied using Saccharomyces cerevisiae nhx mutant strain. The main results are as follows:1.Bioinformation analysis shows that the wheat Na~+/H~+ antiporter TaNHX2 shares higher identity in amino acid sequences with AtNHX1 (70.17%), AtNHX2 (71.95%), OsNHX2 (71.04%), similar proteins from Arabidopsis thaliana and Oryza sativa, which belong to the ClassⅠNHX protein in plant tonoplast. But shares a lower identity with the protein from Lycopersicon esculentum (LeNHX2), only 26.87%, which belongs to the ClassⅡNHX protein in the endosomal. We verified the subcellular localization of the TaNHX2 by using TaNHX2-GFP fusion protein in Arabidopsis protoplasts. The result shows that it is very similar to LeNHX2 which is present in the endosomal membranes such as pre-vacuolar or Golgi membranes. 2. The yeast expression plasmid pYES2-TaNHX2 was constructed and transformed it into yeast mutant strain AXT3K by the polyethylene glycol-lithium acetate method. When TaNHX2 was expressed in AXT3K from vector pYES2, the wheat gene could partly compensate the sensitivity of the yeast strain and grow on AP plates containing 50 mM NaCl or 0.5 M KCl. While the control that the strain only expressed vector pYES2 could not grow on the same stress condition. It demonstrates that TaNHX2 antiporter might not only catalyze Na~+ but also K~+ transport. The Na~+ and K~+ ion contents of transgenic and untransformed yeast mutant strains were measured under 20 mM NaCl stress. Sodium content did not change in transgenic cells as compared to control cells under both normal growth and mild salt stress. On the contrary, the TaNHX2 protein caused the significant increase in potassium content in transgenic cells exposed to 20 mM NaCl treatment. These suggest that the enhanced salt tolerance for transgenic cells is not involved in sodium transport, but potassium accumulation is more important for the tolerance of cells to salt stress. According to the results above, we can say TaNHX2 is a K~+/H~+ antiporter which belongs to the ClassⅠNHX protein and localizes to endomembranes.3. The expression level to different stress of TaNHX2 including NaCl salt stress, low tempture stress at 4oC, ABA ion stress and PEG6000 drought stress was analyzed in the roots and leaves of wheat plants by Real-Time PCR. The result shows that TaNHX2 might function in maintaining the ion balance under normal condition because TaNHX2 expression is observed both in roots and leaves without treatment. Transcript levels of TaNHX2 in roots are induced to a higher level than that in leaves under NaCl salt stress and ABA ion stress, but the expression levels are higher in leaves than in roots under cold stress and drought stress, it showed time-dependant and organ-dependant changes in wheat plants. TaNHX2 play an important role in plants which is affected by abiotic stress or ABA stress, so we can deduce that it may also play a significant role in maintaining cell osmotic balance.4. In order to study the role of the C-terminus in TaNHX2 function, we constructed a series of 6 truncated TaNHX2 versions ranging from the complete one (1614 nucleotides, 538 amino acids) down to the shortest version (1317 nucleotides, 439 amino acids), with no C-termiuns amino acid residue. When TaNHX2-mutant gene and the WT (the complete one) were expressed in AXT3K, there existed difference of the sensitivity of the yeast strain to salt stress between the mutant gene and the WT gene . The result shows that the transgenic yeast transformed 439 mutant gene can survive on plates containing 80 mM NaCl, but can not grow on AP plates containing 0.5 mM LiCl merely, however, the yeast transformed the complete gene can grow on AP plates containing 50 mM NaCl or 0.8 mM LiCl. The OD600 value did not change in transfer-TaNHX2-gene yeast as compared to transfer-439-mutant- gene yeast under 40 mM NaCl when the pH value of the medium is 6.5. On the contrary, the transfer-439-mutant-gene yeast grew better when the pH value of the medium is 4.0. It illustrates the mutant gene can significantly change the sensitivity of nhx mutant strain to salt stress under specific condition.