Molecular Cloning And Functional Identification Of PmNHX1 Gene From Xinjiang Halophyte Plantago Maritima

Salinity is one of the major abiotic stress factors that undermines agricultural production seriously. The excessive sodium ions in soil cause ions toxicity, osmotic stresses, and the damage in K+/Na+ homeostasis. The Na+/H+ antiporters catalyze the exchange of Na+ for H+across the membrane, and they play an important role in the mechanism of plant salt tolerance. The cloning, expression and studying the function of the Na+/H+ antiporter will bring significant meaning to the genetic engineering of plant salt tolerance.Based on the conservative transport protein gene sequence of halophyte, the degenerate primers were designed, and the Na+/H+ antiporter protein gene(2464 bp full-length cDNA) in Plantago maritima at Xinjiang, named as PmNHX1(GenBank accession number: EU233808), was cloned for the first time by RT-PCR and RACE. It was found that the coding sequence of the gene consisted of the 1662 bp nucleotides which encoded 553 amino acids. The molecular weight of the protein was 61.16 kD and the isoelectric point was 7.22. Data analysis showed that the protein mainly located in vacuole membrane including 12 conservative transmembrane domains, TM3 transmembrane domain is "LFFIYLLPPI" - effects of putative amiloride binding domain, and the bit Na+ points and a competitive role, Which was 88%, 89%, 76%, 75%, 73% to sequences of Atriplex gmelini, AgNHX1 (BAB11940), Arabidopsis thaliana, AtNHX1(NP₁87288), Salicornia europaea, SeNHX1(AAN08157), Oryza sativa, OsNHX1(BAA83337), Gossypium hirsutum, GhNHX1 (AAM54141), in amino acid homology respectively.To amplify the ORF sequence of PmNHX1, primers were desinged according to the full length cDNA sequence of PmNHX1. And the PmNHX1 ORF was insert into pMD18-Tsimple cloning vector. The right clone which was ensured by sequencing was used for the constructing of the plant expressional vector pBI121-PmNHX1. The recombinant plasmid pBI121-PmNHX1 was transefered into Agrobacterium tumefaciens(LBA4404) by the liquid nitrogen freezing thaw method.The PmNHX1 gene was transfered into tobacco(Nictiana benthamiana) via agrobacterium mediation. PCR, RT-PCR and Southern blot analysis all showed that the PmNHX1 gene was integrated and transcripted into tobacco genome. Relative electronic conductivity evidently demonstrated less membrane damage in transgenic plants under salt stress compared with the wild type. The transgenic tobacco could survive and grow better than control under 200 mmol?L-1 NaCl treatment.