温度诱导Targetron系统用于大肠杆菌高效基因失活

A temperature-inducible Targetron system for efficient gene inactivation in Escherichia coli

摘要:

构建基于TeI3c/4c嗜热二型内含子的温度诱导Targetron基因失活系统 (Thermotargetron)，并应用于中温微生物基因编辑。在大肠杆菌HMS174 (DE3) 基因组中，选择Subunit of flagellum基因 (fliC) 和C4 dicarboxylate orotate:H+ symporter基因 (dctA) 为靶基因。根据TeI3c/4c DNA识别规则，在fliC和dctA基因中选择fliC489a、fliC828s、fliC1038s和dctA2a位点为基因打靶位点。使用重叠延伸PCR方法，基于pHK-TT1A质粒构建打靶载体。打靶载体转化HMS174菌株，对数期转化子培养液48 ℃热激1 h后涂布于氯霉素抗性LB平板上。使用菌落PCR和DNA测序检测突变株并计算基因失活效率。获得突变株后，通过琼脂穿刺和碳源代谢实验，鉴定ΔfliC、ΔdctA突变株表型变化。菌落PCR测序结果表明，TeI3c/4c插入到fliC和dctA基因设计位点，且打靶效率高达100%。突变株表型验证实验表明，ΔfliC突变株运动能力显著下降，ΔdctA突变株苹果酸代谢能力缺失。综上所述，文中建立了一套适用于嗜中温微生物的温度诱导型、高效基因失活系统，该系统可通过控制宿主菌在48 ℃保温时间实现高效、靶向、精准基因失活。

Abstract:

To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H+ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.