假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与鉴定

根据3种生物的Na^ ／H^ 逆向转运蛋白基因(nhaA)的两端序列设计引物，利用PCR从假单胞菌(Pseudomonas sp．cn4902)中克隆得到一结构基因。该基因长1089bp，编码362个氨基酸，与E．coil K12的nhaA基因的同源性高达97．0％。将该结构基因与pBV220构建成重组载体pBVA。SDS—PAGE电泳表明：含pBVA的转化子产生较高浓度的分子量约为41kD的蛋白，与预期相符。在含NaCl 1．0mol／L的培养基中生长达到平衡期时，转化子的菌浓度约是对照的2．3倍。经原子吸收光谱测定，转化子细胞质中Na^ 浓度仅为对照菌的60．4％。SDS—PAGE电泳表明该基因的表达蛋白位于细胞膜(壁)上。提纯外源基因表达蛋白并对其N端8个氨基酸进行测序，与nhaA基因推测的氨基酸序列完全相符。这些实验证实，克隆得到的基因是假单胞菌的nhaA基因。该基因已经在GenBank登记，收录号为AY643494。

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 1 089 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 ℃ 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.