Isolation and preliminary function analysis of a Na⁺/H⁺ antiporter gene from <i>Malus zumi</i>

Abstract

A full-length cDNA Na+/H+ antiporter gene (MzNHX1) was isolated from Malus zumi according to the homologous Na+/H+ antiporter gene region in plants. Sequence analysis indicated that the cDNA was 2062 bp in length, including an open reading frame (ORF) of 1629 bp, which encoded a predicted polypeptide of 542 amino acids. The MzNHX1 protein shared high identity with other reported plant vacuolar Na+/H+ antiporters. Southern blot analysis detected multiple copies of MzNHX1 in the M. zumi
genome. Northern blot analysis showed negligible expression of the gene in roots, but expression was detected in stems and leaves. To test the function of MzNHX1, we expressed the gene in the saltsensitive
AXT3 yeast mutant. No differences in yeast cell growth were detected given the presence or absence of MzNHX1 on a NaCl free medium. However, on a 70 mM NaCl medium, growth in the control transformant was noticeably suppressed, and yeast overexpression of MzNHX1 showed increased
population growth rates. These results indicated that the MzNHX1 protein increased AXT3 salt tolerance. Alignments of the deduced Na+/H+ antiporter amino acid sequence of different plants from NCBI revealed that MzNHX1 shared high identity (>86%) with vacuolar Na+/H+ antiporters, including RhNHX1 from rose, cNHX1 from citrus, and AtNHX1 from Arabidopsis thaliana. However, MzNHX1 shared very low identity (<10%) with plasma membrane Na+/H+ antiporters such as AtSOS1 from A.
thaliana. These results indicated that MzNHX1 was localized to the vacuolar membrane.