Isolation And Expression Of The Na~+/H~+ Antiporter Gene Of Malus Zumi(MzNHX1) Under Salt Stress

Soil salinization exist all over the world. Salt stress is one of major abiotic stress to plant. Under salt stress, plant injury mainly appears as ion toxicity and osmotic stress. The mainly methods of plants resist salt stress have been found to transport the redundant sodium ions out of cell and compartmentalized them into the vacuole by Na~+/H~+ antiporter. Na~+/H~+ antiporters are membrane proteins and exist in bacteria, yeast, algae, animals and plants widely. They play the major role in maintain osmotic balance, Na~+ homeostasis, the value of K~+/Na~+, and regulate the cytoplast pH, et al. At present, coding Na~+/H~+ antiporter gene sequences which registered in GenBank were more than 400. Malus zumi originated from Japan's northern mountainous area, and was introduced in China in 1976. It has strong resistance, and could grow normally under high salt content. Beyond that, it is an excellent dwarfing rootstock of apple and landscaping tree in saline-alkali soil. Na~+/H~+ antiporter gene of Malus zumi was cloned and expression of gene was analyzed in this study, it has important academic significance.A pair of degenerate primers were designed through the conserved regions of several plants vacuole membrance Na~+/H~+ antiporter nucleotide sequencese in GenBank in this research. The degenerate primers are used for RT-PCR to amplify a cDNA segment from M. zumi. And then the full-length Na~+/H~+ antiporter gene is cloned through RACE method. This gene was named MzNHX1, and its nucleotide sequencese was submitted to GenBank with accession number GQ503257. And it encoding proteins was called MzNHX1 with accession number ADB80440. Sequence analysis shows that the full-length cDNA is 2480bp, and a 1635bp open reading frame encoding a 544 amino acid protein. MzNHX1 amino acid sequence shared highly homology with Arabidopsis thaliana-AtNHX1(AAF21755), Rosa hybrid-RhNHX1(BAD93487.1), Populus euphratica-PeNHX2(ACU01853), and show sequence similarity 78%, 86% and 81% to them, respectively. The MzNHX1 is to belong to the type of NHX-â… , and locate in vacuole membrance. By bioinformatics analyses, we found that the predicted structure of MzNHX1 is similar to Arabidopsis thaliana's, which composed of a hydrophobic N-terminus, 10 transmembrane helix region and 2 non-transmembrane helix region, and a hydrophilic C-terminus. The sequence of 85-LFFIYLLPPI-94 in MzNHX1 TM3 region is highly conserved sequence, binding site of amiloride which is a inhibitor of Na~+/H~+ antiporter. The relative content of NHX gene family were analyzed by through the real-time fluorescence quantitative PCR in M. zumi, M. micromalus, M. sieversii, and the expression of MzNHX1 were also analyzed under NaCl, PEG stress and ABA treatment. The results showed that: highest gene relative content were found in NHX gene family in M. zumi among the three species; next were M. micromalus and M. sieversii, The environmental stresses of salt, drought and ABA could induce the increase of MzNHX1 gene expression, and the gene expression was higher in root than that of in leaves. This demonstrates that MzNHX1 may play a peculiar role in stress resistance of M. zumi. The salt-tolerant molecular mechanisms were revealed preliminary in M. zumi in study. Besides, the research provided excellent genetic resources for breeding salt-tolerant trees, especially the new salt-tolerant of fruit trees through the technique of genetic engineering.