大肠杆菌Zn2+敏感突变株构建及其功能验证

Construction and functional verification of a Zn2+ sensitive mutant of Escherichia coli

摘要:

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用，Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中，zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株，并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株，利用λ Red重组系统，通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上，利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因，获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E.coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件，同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠定了基础。

关键词:基因敲除;λ Red重组;Zn²⁺转运蛋白;zntA;zitB

Abstract:

[Background] Owing to the key roles of Zn²⁺ in cell detoxification and physiological processes, Zn²⁺ transporters have attracted increasing attention. In Escherichia coli, zntA and zitB are two key genes responsible for Zn²⁺ efflux. [Objective] This study aims to construct a Zn²⁺ sensitive mutant of E. coli and verify its function. [Methods] Using E. coli DH5α as the original strain, we knocked out zntA via a λ Red recombination system by using the homologous recombinant fragment carrying a kanamycin resistance gene. On the basis of the single-gene knockout mutant, zitB was further knocked out via the homologous recombinant fragment carrying a gentamicin resistance gene. In this way, a mutant with the knockout of both zntA and zitB was obtained and designated as KZAB04. The functional complementation experiment was conducted to test the sensitivity of the mutants and the original strain (negative control) under different Zn²⁺ concentrations. [Results] KZAB04 exhibited higher Zn²⁺ sensitivity than the original strain. [Conclusion] A Zn²⁺ sensitive mutant of E. coli was successfully constructed. The construction of this mutant is the prerequisite for the functional study of zntA and zitB and lays a foundation for the functional identification of other Zn²⁺ transporter genes.

Key words:gene knockout;λ Red recombination;Zn²⁺ transporter;zntA;zitB