Identification And Characterization Of A Vacuolar Na~+/H~+ Antiporter Gene From Zoysia Japonica L.

Na~+/H~+ antiporter is one of the most valuable gene involving plant salt-tolerance in the world,which catalyzes the exchange of Na~+ for H~+ across membranes rely on the H~+-ATPase or H~+-PPiase proton motive.Na~+/H~+ antiporters also regulate the internal pH,cell volume,sodium level in the cytoplasm and vacuole,and they play an important role in the mechanisms of plant salt tolerance.The studies on cloning, expression,function of the Na~+/H~+ antiporter will bring momentous sense to the genetic engineering of plant salt tolerance.By the multiply alignment of amino acid sequences of several plants vacuole membrane Na~+/H~+ antiporters in GenBank,a pair of degenerate primer was designed against the conserved regions that were used for RT-PCR to amplify a cDNA segment from Zoysia japonica L..Based on the sequences of the cDNA segment,primers were designed for 3'RACE and 5'RACE reactions,and a putative Na~+/H~+ antiporter gene from Zoysia japonica which is a member of the cloned plant NHX-genes family was cloned by RACE method.The gene was named ZjNHX1,and the sequences of ZjNHX1 cDNA have been deposited at GenBank with accession number EU333827. The cloned cDNA of ZjNHX1 is 2421 nucleotides long,with a predicted ORF of 1623 nucleotides,comprising 540 amino acid residues.Multiple alignments revealed a high degree of homology between deduced amino acid sequences of ZjNHX1 and putative vacuolar Na~+/H~+ antiporters of other high plants.The sequence of 'FFIYLLPPI' in ZjNHX1 is highly conserved within other Na~+/H~+ antiporters.The phylogenetic analysis demonstrated that ZjNHX1 formed a cluster with the most closely related plant NHX homolog,which differed from the cluster of plasma membrane Na~+/H~+ antiporter.To test ZjNHX1 function,ZjNHX1 ORF was inserted into the expression vctor pYPGE to construct the yeast expression recombinants pYPGE-ZjNHX1.The construct was transferred into salt-sensitive phenotype of theâ–³nhx1 andâ–³ena1-4â–³nhx1 mutants.It was found that the expression of ZjNHX1 in yeast provides at least partial complementation of the salt-sensitive phenotype of theâ–³nhx1 andâ–³ena1-4â–³nhx1 yeast strains.The results indicated that ZjNHX1 protein plays a similar role with that of the yeast Na~+/H~+ antiporter.The different transcript levels of ZjNHX1 in roots and shoots were examined using real-time PCR method and the result showed that the transcript level of ZjNHX1 in shoots increased 2.45 times after 200mM NaCl treatment,but only slightly increase (1.008-fold) in roots.The analysis of sodium ion content indicated that the plants compartmentalized more Na~+ in shoots compared with wild-type plants.These results implied that ZjNHX1 expression showed a tissue-specific NaCl response pattern.In conclusion,ZjNHX1,cloned from Zoysia japonica L.,functions as the vacuole Na~+/H~+ antiporter,which plays an important role in salt tolerance ion balance in Zoysia japonica L..