Molecular Cloning And Expression Analysis Of A Vacuolar Na~+/H~+ Antiporter Gene From The Succulent Xerophyte Zygophyllum Xanthoxylum

Salinity is one of the major abiotic stress factors that adversely effect the crop plant growth and yield.The excessive sodium ions in soil cause ions toxicity,osmotic stresses, and the damage in K⁺/Na⁺ homeostasis.One of the effective strategies that enable plants to resist salt stress is to compartmentalize Na⁺ into the vacuole by the vacuolar Na⁺/H⁺ antiporter.This process can avert the deleterious effects of Na⁺ in the cytosol and maintain osmotic balance by using Na⁺ accumulated in the vacuole to drive water into the cells,and improve salt- and drought- tolerance of plants.Therefore,it is important meaning for genetic improvement of salt- and drought- tolerance of the crops and herbages to isolate and characterize the vacuolar Na⁺/H⁺ antiporter from the desert plants lived in extremely arid environment.In this study,the vacuolar Na⁺/H⁺ antiporter gene,ZxNHX,and the fragment of actin gene were cloned from the succulent xerophyte Zygophyllum xanthoxylum;and expression of ZxNHX was analyzed using actin as internal control under salt stress.The main results were as following:1.Degenerate primers were designed based on the conserved sequences of the vacuolar Na⁺/H⁺ antiporter genes from other plants.A novel vacuolar Na⁺/H⁺ antiporter gene was isolated from Z.xanthoxylum using RT-PCR and RACE,and named as ZxNHX.The cDNA is 2127 bp in length including an open reading frame(ORF)of 1558 bp,a 5'untranslated region(5'URT)of 213 bp and a 3'untranslated region(3'URT)of 315 bp with a ploy(A) tail.The ZxNHX cDNA encodes a protein of 532 amino acids with a deduced molecular mass of 58.8 KDa and a pI of 7.23.The ZxNHX protein shares 81%,80%,79%,78%,74% and 73%homology with the six reported other NHXs,namely PeNHX2(DQ414512), AtNHX1(AY685183),PtNHX1(AY660749),AgNHX1(AB038492),SsNHX1(AF370358) and OsNHX1(AY324877),respectively.ZxNHX has twelve putative transmembrane(TM) domains,the TM3 of which contains the conserved amiloride-binding sites (⁷⁸LFFYLLPPI⁸⁷).In addition,two potential N-glycosylation sites were found within the ZxNHX protein,at Asn⁴⁴and Asn²⁸⁷,respectively.These results suggested that the ZxNHX protein is glycosylated.The gene has been submitted to GenBank database,the accession number is EU103624.2.Degenerate primers were designed based on the conserved sequences of the actin genes from other plants.The fragment of actin gene was isolated from Z.xanthoxylum by RT-PCR,and named as ZxACT.The sequencing result revealed that the actin gene fragment from Z.xanthoxylum is 598 bp in length,encoding a protein of 198 amino acids. Homology comparison with other plants actin gene sequences in the GenBank shows that it shares over 82%nucleotide sequence homology and 91%amino acid sequence homology with other plants actins.The gene has been submitted to GenBank database,the accession number is EU019550.3.Semi-quantitative RT-PCR analysis results showed ZxNHX was expressed strongly in leaves while weakly in roots.The transcript level of ZxNHX in the leaves was up-regulated by 150 or 200 mmol·L^-1NaCl,and highest at 3 or 12 h.These results suggested the expression of ZxNHX was induced and regulated by salt stress.