Isolation, Functional Characterization And Expression Pattern Of A Vacuolar Na~+/H~+ Antiporter Gene FtNHX From F. Tartaricum Gaertn

Salt injury is a major limited factor for plant growth and production. It is a very important research filed of understanding the mechanism of plant salt-tolerace, cloning salt-tolerant gene and cultivating new varieties of high salt-tolerant plant.Salt stress mainly caused by Na+ions. High Na+concentration leads to ion toxicity, osmotic stress, K+/Na+ratio imbalance, abnormal growth and metabolism and then causes significant damage to plants. Na+/H+antiporter, dependent on the energy generated from hydrolysis of ATP or H+ electrochemical gradient, catalyzes the exchange of Na+ and H+ ions, which is a major mechanism of plant salt tolerance. Study on the cloning Na+/H+ antiporter genes from different plant species, analyzing the structural and functional relations of these genes will be valuable for further understanding the molecular mechanism of plant salt tolerance and molecular breeding of salt tolerant plants. In this research project, we firstly cloned a Na+/H+ antiporter gene from F.tataricum Gaertn and studied its fuction and expression patterns.A vacuolar Na+/H+ antiporter of F.tataricum Gaertn was isolated and cloned by RT-PCR and RACE method. The cDNA is 2052bp in length, including 1662bp ORF (Open Reading Frame),287 bp 5'NTR (Non Translated Region),75 bp 3'NTR and 28 bp polyA tail. This gene is named FtNHX, and its GeneBank accession number is GU984045. The predicted ORF encodes a protein of 553 amino acids with a calculated molecular mass of 61 KDa.The bioinformatic analysis shows that FtNHX has the typical structure features of the Na+/H+ antiporter. It contains a highly conserved LFFIYLLPPI site, a hydrophobic N-terminal,8 transmembrane regions and a C-terminal hydrophilic region for regulatory function. Alignment and phylogenetic analysis demonstrated that FtNHX shares high identities (above 50%) to IC-NHE proteins such as AtNHX1-4 and ZmNHX1, which were located in tonoplast. However, FtNHX shares lower identitis (less than 10%) to PE-NHE proteins such as OsSOS and PeSOS, which belong to the plasma-type Na+/H+ antiporter. The data implied that FtNHX encoding product is a vacuole-type Na+/H+ antiporter.The yeast complementary experiment was conducted to test the function of FtNHX. We have successfully constructed yeast expression vector pYPGE15-FtNHX, and transformed pYPGE15-FtNHX into a salt-sensitive mutant yeast strain (△ enal-4△nhx). The results showed that overexpression of FtNHX in△enal-4△nhx yeast strain caused growth recovery of the mutant in APG medium with 100mM NaCl, 1.5M KCl,2.5mM LiCl respectively and YPD medium with 50μg/mL HYG. The test results indicated that FtNHX work as Na+/H+ antiporter and plays an important role in salt tolerance.Real-Time PCR were preformed to analyze the transcript level of FtNHX in buckwheat different tissues after salt stress. The result showed that the transcript levels of FtNHX, after 100 mM NaCl stress, increased 1.15-fold in roots,1.31-fold in stems and 3.16-fold in leaves comparing to the controls, respectively. These results suggested that FtNHX expression had showed a tissue-specific NaCl response pattern.