Study On Cloning Of CDNA Encoding Na～+/H～+ Antiporter From Tonoplast Membrane Of Aeluropus Littoralis And Salt Tolerance Of Its Transgenic Tobacco

Salt stress is mainly caused by Na~+. The Na~+/H~+ antiporters catalyze the exchange of Na~+ for H~+ across the membrane, and they play an important role in the mechanisms of plant salt tolerance.The cloning, expression and studying the function of the Na~+/H~+ antiporter will bring momentous sense to the genetic engineering of plant salt tolerance.In order to isolate the full length cDNA of Na~+/H~+ antiporter gene from the tonoplast membrane of Aeluropus littoralis, RT-PCR and RLM-RACE were carried out, and a pair of degenerate primers were designed according the totonoplast Na~+/H~+ antiporter homologous gene region of other plants. The gene was named AlNHX1. AlNHX1 cDNA (2706bp) included a 387bp 5' UTR, a 696bp 3' UTR, a putative polyadenylylation signal and a 1623bp open reading frame encoding a 540-amino-acid polypeptide which was 94 %, 90 %, 88 %, 89 %, 75 %, 76 %, 75 %, 73 % to sequences of Phragmites australis, Oryza sativa, Triticum aestivum, Hordeum vulgare, Zea mays , Atriplex gmelini, Salicornia europaea Arabidopsis thaliana, in amino acid homology respectively. The deduced amino acid sequence included the conserved amiloride binding sites of LFFIYLLPPI. A was considered as the possible transcription initiation site based on the principle of RLM-RACE and the analysis of AlNHX1 5' UTR sequences, and it would establish foundation for the studying of its relating and expression.To amplify the ORF sequence of AlNHX1, Primers were designed according to the full length cDNA sequence of AlNHX1. And the AlNHX1 ORF was insert into the plasnid of pUC19. The right clone which was ensured by sequencing was used for the constructing of the plant expression vector pBI121-NHX. The resultant plasmid pBI121-NHX was transferred into Agrobacterium tumefaciens (GV3101) by the liquid nitrogen freezing thaw method.