Cloning And Characterization Of Na～+/H～+ Antiport Genes From Salicornia Europaea L. And Tetragonia Tetragonioides O.

Salt stress is mainly caused by sodium ions. High concentration of sodium ions lead to ion toxiciry, osmotic stress and K+/Na+ ratio imbalances, then result in deleterious effect on critical biochemical process. The NaVhT antiport catalyzes the exchange of Na+ for H+ across the membranes, and plays an important role in mechanisms of plant salt tolerance. Cloning and expression of NaVH* antiport gene, understanding of the structure and function of the antiport facilities the research on salt resistant mechamism at molecular level. For this purpose molecular cloning of Na+/H+ antiport genes from Salicornia europaea L. and Tetragonia tetragonioides O. were performed in this thesis research.In order to isolate the full length cDNA of SeNHXl gene, RT-PCR, 3' RACE and 5' RACE were carried out, and the degenerate primers were designed according to the homologous gene region. The cDNA is 2668bp in length including an open reading frame of 1683bp, 491bp 5' UTR(untranslated region), 443bp 3' UTR and 51bp Oligo d(T) tail. The SeNHXl cDNA encodes a predicted polypeptide of 560 amino acids, with a predicated molecular mass of 62.1KDa. The SeNHXl protein shares 88%, 74%, 77%, 78%, 63% identity with the five other reported NHXs, namely AgNHX1(ABQ38492), OsNHX1(AB021878), AtNHX1(AY040245), cNHX1(AY028416), TaNHX1(AY040245), respectively. Southern blotting analysis showes that one copy of SeNHXl is in the Salicornia europaea genome. The expression patterns were analyzed by RT-PCR. SeNHXl transcripts are detected in shoots under normal growth condition, but increases after NaCl and ABA treatments. But in roots none SeNHXl transcripts can be detected under either condition. The gene has been submitted to the GenBank database, and the accession number is AY131235; and applied for patent, the apply number is 03105029.8.A Na+/H+ antiport full length cDNA was cloned from Tetragonia tetragonioides by RT-PCR and RACE strategy, and named TtNHX1 The cDNA is 2199bp in length including an open reading frame of 1665bp, 91bp 5' UTR, 413bp 3' UTR and 31bp Oligo d(T) tail. The SeNHXl cDNA encodes a predicted polypeptide of 552 amino acids, with a predicated molecular mass of 59.5KDa. The TtNHXl protein shares 76%. 61%, 70%, 65%, 61% identity with the five other reported NHXs, namely AgNHXl(AB038492), 0sNHX1(AB021878),AtNHX1(AY040245), cNHX1(AY028416), TaNHX1(AY040245), respectively. Southern blotting analysis showes that two or three copies of TtNHXl are in the Tetragonia tetragonioides genome. Northern blotting analysis showes that TtNHXl gene expresses in roots, stems and leaves, no expression is detected in flowers. TtNHXl gene has been submitted to the GenBank database, and the accession number is AF527625; and applied for patent, the apply number is 03109436.8.SeNHXl and TtNHXl gene has high identity of 71%, SeNHXl protein shares 85% identity with TtNHXl protein. 12 predicted transmenbrane domains are detected in SeNHXl and TtNHXl protein, with high similarities. The sequence of LFFIYLLPPI in SeNHXl and TtNHXl is highly conserved, this region is the binding site of amiloride. SeNHXl -, TtNHXl is a vacuolar-type Na+/H+ antiport gene, and induced by salt stress.