解纤维梭菌脱水四环素诱导的ClosTron基因打靶系统构建

Construction of anhydrotetracycline-inducible ClosTron system in Clostridium cellulolyticum

摘要:

[背景] 解纤维梭菌是发酵木质纤维素生产乙醇的中温模式菌株，构建可控诱导的基因打靶技术是研究解纤维梭菌遗传调控的重要手段。[目的] 在解纤维梭菌中构建脱水四环素诱导的ClosTron基因打靶系统。[方法] 首先，分析解纤维梭菌对脱水四环素的敏感性，筛选合适的诱导剂浓度，并以β-葡萄糖苷酶为报告基因，检测其在解纤维梭菌中的诱导效果。然后，将脱水四环素诱导型操纵子与II型内含子元件组合，构建脱水四环素诱导的ClosTron质粒。最后，以解纤维梭菌mspI、ldh和ack为例，检测其在解纤维梭菌中的打靶效率。[结果] 脱水四环素诱导系统在解纤维梭菌中能够诱导ClosTron元件表达，在mspI、ldh和ack这3个基因位点的打靶效率分别为29.48%±15.51%、23.61%±7.08%和28.09%±6.97%。[结论] 在解纤维梭菌中成功构建脱水四环素诱导的ClosTron基因打靶系统，为梭菌基因工程改造奠定了基础。

Abstract:

[Background] Clostridium cellulolyticum is a mesophilic model strain for ethanol production from lignocellulose. Constructing the gene targeting technology with controllable induction is an important means to study the genetic regulation of C. cellulolyticum. [Objective] To construct an anhydrotetracycline-inducible ClosTron gene targeting system in C. cellulolyticum.[Methods] Firstly, we determined the sensitivity of C. cellulolyticum to anhydrotetracycline and selected the appropriate inducer concentration, and then tested the inducible effect using β-glucosidase gene as reporter gene. Secondly, we constructed an anhydrotetracycline-induced ClosTron plasmid by combining the anhydrotetracycline-inducible operon with group II intron elements. Finally, the mspI, ldh and ack were selected as targets to determine the targeting efficiency in C. cellulolyticum.[Results] The anhydrotetracycline-inducible system could induce the ClosTron expression in C. cellulolyticum, and the targeting efficiency at mspI, ldh and ack sites were 29.48%±15.51%, 23.61%±7.08% and 28.09%±6.97%, respectively. [Conclusion] The anhydrotetracycline-inducible ClosTron system was successfully constructed in C. cellulolyticum, which laid a foundation for genetic engineering of Clostridium.