铜绿假单胞菌中快速基因操作工具的开发和应用

Development and application of a rapid gene manipulating toolbox for Pseudomonas aeruginosa

针对基因的操作,包括缺失和插入基因、替换基因元件(如启动子)、融合荧光蛋白基因、构建原位的基因报告系统,是大多数生物技术实验室的必备技术。目前广泛使用的基于2次同源重组的基因操作方法,在构建质粒、转化和筛选方面较为繁琐。另外使用该方法进行长片段敲除的效率较低。为了简化基因操作的流程,本研究构建了一个最小化的铜绿假单胞菌(Pseudomonas aeruginosa)整合型质粒pln2,只需将目标基因内部一段序列克隆到pln2质粒并导入细菌,由于质粒不能在细菌内自我复制而只能通过单次等位基因交换整合到基因组上,从而使目的基因断裂为两部分而失去活性。在pln2的基础上开发了一整套工具质粒适用于基因组的不同操作,包括融合荧光蛋白基因、替换基因元件(如启动子)、构建原位的转录型荧光报告系统。此外,本研究借助该系统成功实现超长片段基因簇的敲除,单次可以敲除长达270 kb的片段。

Manipulation of genes,including knock-out or knock-in,replacement of gene elements(such as promoters),fusion with a fluorescent protein gene,and construction of in situ gene reporter,is required in most of the biotechnological laboratories.The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids,transforming and screening.In addition,the efficiency of using this method for long fragment knockout is low.To simplify the process of gene manipulation,we constructed a minimized integrative vector pln2.When a gene needs to be inactivated,an internal fragment of the target gene(non-frameshift)is cloned into the pln2 plasmid.Once the single-crossover recombination between genome and the constructed plasmid occurs,the endogenous gene is segmented by the plasmid backbone and thus inactivated.We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above.With the help of this toolbox,we successfully knocked out large fragments of 20–270 kb.