假单胞菌Na^+/H^+逆向转运蛋白基因nhaA的克隆与鉴定  

Cloning and Characterization of Na^+/H^+ Antiporter Gene (nhaA) from Pseudomonas sp.cn4902

作　　者：刘广发[1] 曾活水[2] 陈启伟[1] 高亚辉[1] 

机构地区：[1]厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005 [2]国家海洋局第三海洋研究所,厦门361005

出　　处：《Acta Genetica Sinica》2005年第3期309-314,共6页

基　　金：福建省科技计划重点资助项目(编号:2003N053);厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室项目(编号:2004106)~~

摘　　要：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 (Pseudomonassp.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.coliK12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NaCl1.0mol/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。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.

关 键 词：蛋白基因 NA^+ 蛋白 对照 克隆 逆向转运 假单胞菌 转化子 外源基因表达 SDS—PAGE 

分 类 号：Q785[生物学—分子生物学]