[Cloning and characterization of Na+ / H+ antiporter gene (nhaA) from Pseudomonas sp. cn4902]

According to the sequences of the gene nhaA coding for Na+ / H+ antiporter,a structural gene was cloned from Pseudomonas sp. cn4902 by PCR reaction with a set of primers. It was 1089 bp in length and codes for 362 amino acids sharing homology with the gene nhaA of E. coli K12 as high as 97.0%. It was inserted into plasmid pBV220 to form a high level expression reconstruction plasmid pBVA. So an overexpression 41 kD protein band could be found in the lane of transformant harbored with pBVA after SDS-PAGE electrophoresis. The detection of growth curve showed that the biomass of the transformant was 2.3 times over that of the control in the medium containing 1.0 mol/L NaCl. It was found that Na+ concentration in cytoplasm of the transformant was low to 60.4% of the control by the detection of atomic absorption spectrum. Evidence of SDS-PAGE electrophoresis of membrane proteins also showed that the NhaA was located in membrane. Purified NhaA was harvested and digested by FXa proteinase. The sequence of eight amino acids in N termination of NhaA protein was entirely identical with the polypeptide deduced from the nhaA gene. Then ten strains of transformant were continuously cultivated for 18 generations under 42 degrees C hot shock condition,all of their reconstructed plasmids were lost with the result that salt-tolerant-level went back to the original standard. In summary, all the experiments proved that the cloned gene is nhaA gene. The gene has been accepted in GenBank by the accession number AY643494.