双启动子级联调控荧光蛋白基因表达载体的构建及环境监测应用

A variety of regulatory elements responded to pollutants in microbial genomes can be used to build the genetically engineered bacteria for environmental monitoring, which has broad application prospects in the detection of pollutants as well as its toxic effects. During the screening and construction of microorganisms used in environmental monitoring, the sensitivity, accuracy and simplicity have been the key points. In this study, specific promoter responded to pollutants will be connected with the powerful T7 promoter by the way of cascade regulation to construct a fluorescent protein gene expression vector regulated by two cascade promoters, in which the T7 promoter will be driven by the start-up of specific promoter, and fluorescent protein gene under the control of T7 promoter will be expressed. The recombinant expression vector will be transferred to suitable host bacteria to build genetically engineered bacteria with high sensitivity to environmental contaminants, which application in environmental monitoring will be also researched. The sensitivity of the genetically engineered bacteria to the pollutants could be raised by amplifying expression of the reporter gene through the cascade regulation; the successful establishment of this model will provide a new approach and ideas to promote sensitivity of genetically engineered bacteria to environmental pollutants, which will extend the application prospect of genetically engineered bacteria in environmental monitoring.

利用微生物基因组中对污染物有响应的多种调控元件可以构建用于环境监测的基因工程报告菌，在污染物的监测以及毒性效应检测等方面具有广阔的应用前景。在筛选和构建这类用于环境监测的微生物菌株时，灵敏性、准确性和简便性一直是研究的重点。本研究拟通过级联调控方式，将对污染物有响应的特异启动子与强效表达启动子T7关联起来，通过特异启动子开启强效T7启动子，再由T7启动子启动报告基因荧光蛋白基因的表达，构建出一个双启动子级联调控的荧光蛋白表达载体，并将该载体转入合适的宿主菌中，构建出对环境污染物高敏感性的基因工程报告菌，并进行环境样品监测应用研究。该模式是通过放大报告基因的表达来提高基因工程菌对污染物的灵敏性，这种模式的成功建立将为提高基因工程菌对环境污染物的灵敏度提供一种新方法和新思路，并推动基因工程菌在环境监测领域的应用研究。

利用微生物基因组中对污染物有响应的多种调控元件可以构建环境监测用的基因工程报告菌，在污染物的监测以及毒性效应检测等方面具有广阔的应用前景。在这类基因工程菌的筛选和构建中，启动子和报告基因是关键因素，启动子决定了基因工程菌对于环境污染物的敏感性和特异性，选用的报告基因决定了检测的简便性。本项目在研究中利用基因重组技术成功构建了一个双启动子级联调控荧光蛋白表达载体，在该载体中将对污染物砷有响应的特异启动子（Pars）与强效表达启动子T7关联起来，通过砷特异启动子开启强效T7启动子，再由T7启动子启动报告基因绿色荧光蛋白基因的表达，这种级联调控方式在实验中证实可以放大报告基因的表达并对诱导剂表现出更快、更高的响应性。在实验室条件下，利用该载体构建的基因工程报告菌的荧光强度与低浓度的砷诱导剂之间的呈现出良好线性关系。这种双启动子级联调控模式的成功建立能够会为提高基因工程菌对环境污染物的灵敏度提供一种新方法和新思路，并且还可能会为基因表达调控研究提供新的技术便利。