Isolation, Functional Characterization, and Expression Pattern of a Vacuolar Na+/H+ Antiporter Gene TrNHX1 from Trifolium repens L.

Plant vacuolar Na+/H+ antiporter plays an important role in salt tolerance. A vacuolar Na+/H+ antiporter gene TrNHX1 was cloned from Trifolium repens L., a forage legume, by RT-PCR and RACE methods using degenerate oligonucleotide primers. The TrNHX1 sequence contains 2,394 nucleotides and an open-reading frame of 1,626 nucleotides that encodes a protein of 541 amino acids with a deduced molecular mass of 59.5 kDa. The deduced amino acid sequence of TrNHX1 is 78% identical to that of a vacuolar Na+/H+ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. TrNHX1 could partially complement the NaCl-sensitive phenotypes of yeast mutants Delta nhx1 and Delta ena1-4 Delta nhx1, and a similar complementation was also observed in the presence of LiCl and KCl. In addition, it was found that TrNHX1 suppressed the hygromycin-sensitive phenotype of yeast mutant Delta ena1-4 Delta nhx1. The expression of TrNHX1 in T. repens increased in the presence of 150 mM NaCl, and this result accords with that of Na+ contents determination under the same treatment. These results suggest that TrNHX1 functions as a vacuolar Na+/H+ antiporter and plays an important role in salt tolerance and ion homeostasis in T. repens.