大肠杆菌中dctA基因敲除及其苹果酸摄取功能鉴定  

Knock-out of dctA gene from Escherichia coli K12 and characterization of its function for malic acid transportation

作　　者：姜巨全[1] 张正来 徐桐 孟琳 JIANG Juquan;ZHANG Zhenglai;XU Tong;MENG Lin(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2018年第5期45-52,共8页Journal of Northeast Agricultural University

基　　金：国家自然科学基金项目(31770051;31570087)

摘　　要：大肠杆菌(Escherichia coli)K12基因组中,仅dctA基因编码1个在有氧条件下负责摄取四碳-二羧酸的转运蛋白。为鉴定Dct A能否摄取四碳-2-羟基羧酸(如苹果酸),首先以p KD3为模版PCR扩增含有氯霉素抗性dctA基因同源臂,电转入E.coli K12/p KD46感受态细胞,筛选具有氯霉素抗性的基因敲除突变株E.coliΔdctA/p KD46。通过消除质粒p KD46构建大肠杆菌dctA基因敲除突变株E.coliΔdctA。构建dctA基因表达载体p Easy T3-dctA。通过苹果酸摄取功能互补试验,证实基因敲除菌株E.coliΔdctA丧失摄取苹果酸功能,四碳-二羧酸摄取蛋白Dct A具有摄取苹果酸功能。研究首次报道Dct A具有摄取苹果酸功能,该基因敲除突变株的构建可为其他菌株来源2-HCT转运蛋白的基因克隆与功能分析奠定基础。In Escherichia coli K12, dctA is the sole gene that encodes a aerobic transporter for uptaking C4-dicarboxylic acids. However, it had not been reported by domestic researchers whether it was capable of taking up C4-2-hydroxycarboxylic acids, such as malic acid. For the characterization of DctA as a C4-2-hydroxycarboxylic acid transporter, a chloramphenicol resistance gene cmR was amplified by PCR to contain 50-nt homologous arms with dctA gene from E. coli using the template plasmid pKD3 and then electroporated into electrocompetent cells of E. coli K12 with pKD46. The dctAdeficient E. coli mutant with pKD46, ΔdctA /pKD46, was obtained by screening colonies with chloramphenicol resistance. Finally, dctA-deficient E. coli mutant ΔdctA succeeded to be constructed after pKD46 was eliminated from E. coli ΔdctA / pKD46. Also, the expression vector of dctA gene designated pEasy T3-dctA was constructed. The growth tests for uptaking malic acid were carried out through functional complementation. The results revealed that dctA-deficient E. coli mutant ΔdctA lost the ability of uptaking of malic acid and dctA gene was indeed capable of taking up malic acid. This was the first report on the function of DctA as a transporter for uptaking of malic acid. The successful construction of E. coli ΔdctA was very helpful for cloning and characterization of heterogenous genes encoding transporters for uptaking of C4-2-hydroxycarboxylic acids by functional complementation.

关 键 词：大肠杆菌K12 dctA λRed重组 基因敲除 四碳-2-羟基羧酸 

分 类 号：Q933[生物学—微生物学] Q751