Isolation and molecular characterization of a novel Na + /H + antiporter gene, AlNHX2, from Aeluropus littoralis and comparison of AlNHX1 and AlNHX2

Abstract

Halophytes have high tolerance against salinity and it is expected that these plants have special proteins that allow them to thrive under salinity conditions. Hence, to understand molecular aspects of vacuolar Na + /H + antiporter, which has a possible role in salt tolerance in halophytic plants, a novel Na + /H + antiporter gene, AlNHX2, was isolated and characterized by rapid amplification of cDNA ends (RACE) technique. The results revealed that AlNHX2 is expressed in leaves, stems and roots and its expression in leaves is 1.767 and 1.269 times higher than stems and roots, respectively. This gene has an ORF with 1617 bp in length, a 3'-UTR region with 245 pb, and a 5'-UTR region with 187 bp which encodes a 538 amino acid protein shared a high homology with those putative vacuolar Na + /H + antiporters of higher plants. Putative phosphorylation sites within AlNHX1 and AlNHX2 were determined using the prediction software, and the binding of 14-3-3 protein to specific domains within AlNHX1 and AlNHX2 was predicted. The results also revealed this binding is induced by a protein kinase-mediated phosphorylation of a specific Thr or Ser residue in these domains. The results also revealed that the activities of AlNHX1 and AlNHX2 are regulated by PKC, p38MAPK and GSK3. In addition, the findings reported here show the interaction of CaM protein with C-terminal of AlNHX2. Autoinhibitory domain at C-terminal region of AlNHX2 that can suppress the protein activity under normal growth conditions was also found.