迪茨氏属菌的石油降解基因及调控机制研究

项目名称： 迪茨氏属菌的石油降解基因及调控机制研究

项目编号： No.31200099

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 微生物学、植物学

项目作者： 卢社莲

作者单位： 北京大学

项目金额： 22万元

中文摘要： 本项目拟在本实验室分离纯化的迪茨氏属菌Dietzia sp.DQ12-45-1b 工作的基础上，进一步深入研究石油降解基因alkB和CYP450家族（CYP153是其中一个亚型）基因在石油降解中的协同功能及调控机制。通过分子克隆技术构建alkB和CYP450家族基因的高表达菌株及alkB和CYP450功能缺陷菌株，之后观察不同菌株型对烷烃的石油降解功能；通过SI核酸酶保护实验和primer extension实验，报告基因，RNase保护实验，分析Dietzia sp.DQ12-45-1b中alkB和CYP153基因簇的转录起始位点，转录调控模式和机制；比较alkB和CYP153的转录调控方式，阐明alkB和CYP153在调控水平的协同机制。alkB和CYP450基因功能研究的深入, 将为三次石油生物开采和环境石油污染的生物修复工程菌开发提供理论依据。

中文关键词： Dietzia；单同源臂线性DNA；基因敲除；转化效率；正交实验

英文摘要： It is reported that alkB and cytochrome P450 (CYP153) genes can be cloned from many oil-degradation bacteria. Meanwhile, there studies mostly focus on the gene detection, but the concrete functional regulation mechanism is still not clear. The aim of present study is to further research the function and regulation mechanism of Dietzia sp.DQ12-45-1b key oil-degradation alkB and CYP450 family (including 13 subtypes ) genes based on the work that we have done in our lab. To achieve this goal, the Dietzia sp. DQ12-45-1b alkB and CYP450 genes over-expression by transformation alkB-expressed plasmid into bacteria and low expression by group II intron to insert themselves ef?ciently into virtually desired DNA target site via the activity of an RNA-protein complex (RNP) will be construct by molecular and gene knockout technology. Then detect the oil-degradation capability of different types bacteria. AlkB and CYP153 transcriptional start site of Dietzia sp. DQ12-45-1b will be analyzed by SI nuclease protect experiment and primer extention experiment. The alkB and CYP153 gene cluster transcriptional control model and protein expression control will be study by reporter genes and DNase protect experiment .The further research of alkane hydroxylase alkB and cytochrome P450 genes will contribute to construct engineering ba

英文关键词： Dietzia；mono-homologous arm linear DNA；gene knockout；transformation efficiency；orthogonal experimental design