一种基于lacZ基因和pUC复制子的原核启动子报告系统的构建及评价

Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene

摘要:

为构建一种具有广泛适用性的原核启动子报告系统，以质粒pFLX107为骨架，通过多克隆位点替换和序列改造，构建出基于lacZ基因和pUC复制子的pFGH系列报告载体，然后以lacZ基因缺失株MC4100为宿主菌筛选背景活性最低的质粒作为最终的报告系统，并利用诱导型启动子araBAD和组成型启动子rpsM分别对其进行测试。结果显示，在所构建的pFGH系列质粒中，pFGH06的背景活性显著低于同系列其他质粒，在28 ℃培养条件下甚至显著低于低拷贝参考质粒pRCL的活性 (P<0.01)。进一步的评估测试显示，质粒pFGH06可用于诱导型启动子或组成型启动子的克隆及活性测定，且在模拟应用于启动子筛选时，通过蓝白斑筛选即可实现对目标启动子的完全识别。与已报道的原核启动子报告系统相比，pFGH06具有体积小、克隆位点多、背景活性可调、对启动子筛选识别效率高等优点，具有广泛的应用前景。

Abstract:

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.