Cloning, Expression And Genetic Transformation Of A Vacuolar Na~+/H~+ Antiporter Gene In Galega Orientalis

High concentrations of salt in soils account for large decreases in the yield of a wild variety of crops all over the world.Plant salt-tolerant genetic engineering was considered as the most effective and economical mean to bring up salt-tolerant crops and to exploit salt wasteland.In plants,by the energy provided by transporting proton along its concentration gradient,vacuolar-type Na~+/H~+ antiporters deliver Na~+ into the vacuoles against the electrochemical gradient generated by vacuolar H~+-ATPase and H~+-PPiase.This compartmentation of Na~+ provides an effective mechanism to avert the deleterious effects of Na~+ in cytosol and maintain the normal K~+/Na~+ ratio,and an osmotic potential by using Na~+ in the vacuoles as an osmoticum.In this study,a full length cDNA of NHX1 gene in Galega orientalis(GoNHX1) was cloned using RT-PCR and RACE,with primers designed according to the sequence of CkNHX1.The data shown that the GoNHX1 gene has 2280bp in length,including an open reading frame of 1626bp which encoded a protein of 542 amino acid residues.The estimated molecular weight and isoelectric points of the putative protein was 60.0 kDa. Components of amino acids encoded by GoNHX1 contained 41 basic amino acids,35 acidic amino acid,231 hydrophobic amino acids and 144 polar amino acids.The predicted secondary structure composition for the protein has about 44.18%alpha helixes,19.96% extended strand,5.36%beta turn and 30.50%random coil,10 potential transmembrane segments.Phylogenetic analysis showed that GoNHX1 share the same group with vacuolar-type Na~+/H~+ antiporter,and distinguished from plasma membrane-type Na~+/H~+ antiporter.RT-PCR results revealed that GoNHX1 was belonging to inducible expression, which was expressed in the roots,stems and leaves in NaCl stress.GoNHX1-GFP fusion protein was constructed and transiently expressed in onion epidermal cells.The green fluorescence was detected on the tonoplast,demonstrating that GoNHX1 subcellular localization on vacuolar membrane.GoNHX1 expression vector pCAMBIA1300 were constructed and introduced into Tobacco plants.The result of PCR showed that the GoNHX1 gene cDNA had transfer-into the Tobacco.