Cloning And Characterization Of Tonoplast Na~+H~+Antiporter From Nitraria Sibirica Pall

The Nitraria L. is a small genus of Zygophyllaceae, which primarily grows in arid and semiarid regions, saline and alkali land, desert and sandy areas, and is the major constructive species of forest in western Inner Mongolia. The Nitraria plants exhibit a potent salt-resistance, and have important economical-value and ecological-value. Therefore, the research on molecular mechanism of stress tolerance is especially necessary in Nitraria.Tonoplast Na+/H+antiporters play an important role in plants salt tolerance, which can deliver Na+into the vacuoles against the electrochemical gradient. The compartmentation of Na+provides an effective mechanism to avert the deleterious effects of Na+on cytosol, and maintain an osmotic potential by using Na+in the vacuoles as an osmoticum.In the study, using the Reverse Transcription (RT)-PCR and rapid amplification of cDNA ends (RACE) methods, a Na+/H+antiporter gene, referred to NsNHX1, was isolated from a halophytic plant, Nitraria sibirica. The cloned NsNHX1cDNA contains2182nucleotides, with a predicted ORF of1635nucleotides, a5’-untranslated region of148nucleotides, and a3’-untranslated region of399nucleotides. The predicted ORF encodes a protein of544amino acids, which has high homology with ZxNHX (Zygophyllum xanthoxylum) and CpNHX(Citrus x paradisi).Semi-quantitative RT-PCR analysis shows that the mRNA level of NsNHX1is significantly higher in leaves than those in stems or roots. The steady-state level of NsNHX1transcript is up-regulated by treatment with NaCl, cold or ABA, and not induced by drought stress. This result is consist with some regulatory and cis-acting elements existed in a807bp of the upsteam region adjacent to the NsNHX1, which was isolated by the thermal asymmetric interlaced TAIL-PCR. The result suggests that the NsNHX1expression can be up regulated by salt, cold and exogenous ABA. Copy numbers of NHX gene in the N. sibirica genome were confirmed by DNA gel blot analysis. The results indicated that there were two or three copies of NsNHX1gene in N. sibirica genome. Transient expression of an NsNHXl-GFP translational fusion product in onion epidermal cells is visualized by epifluorescence microscopy. GFP fluorescence is concentrated on the vacuolar membrane. The result indicates that the NsNHX1is located in tonoplast of cells.To analyze the function and expression of NsNHX1, the transgenic Arabidopsis with heterologous expressed of NsNHX1was constracted. Salt-resistance analysis of T3homozygous plants indicated that the transgenic Arabidopsis has obvious superiority in germination, and growth of roots and seedlings under salt stress condition. These results indicated that the expression of NsNHXl could improves salt-resistance of transgenic plants. These results would provide theoretical and practical proof for the salt tolerance genetic improvement of plants.