红平红球菌KB1低温降解烷烃关键基因的确定及调控机制研究

利用微生物为主体的生物修复技术是降解环境中石油烃的主要途径，已成为当今环境科学研究的热点。本项目以从甘肃玉门油田分离的一株在低温下具有高效石油烃降解能力的红平红球菌KB1为研究对象，拟采用全基因组测序技术获取完整基因组信息，借助生物信息学方法分析其潜在的烷烃氧化酶基因、基因簇结构和转录调控因子，结合基因突变技术和代谢组学技术研究烷烃氧化酶基因的功能和代谢机制；同时利用DNA亲和纯化技术进一步筛选新的转录调控因子，凝胶阻滞实验验证烷烃氧化酶基因启动子序列与转录调控因子间的相互作用，进一步构建转录调控因子基因的缺失菌株，研究其低温下的石油烃降解调控机制。该研究结果可为充分挖掘微生物功能基因资源，诠释微生物降解石油烃的分子机制和设计高代谢活性的基因工程菌株等提供新的研究思路，具有重要的理论研究和实际应用价值。

Microbial bioremediation is the main pathway to degrade petroleum hydrocarbons in the environment, and has been the focus of environmental science research. In the study, a strain of Rhodococcus erythropolis KB1, which was isolated from Yumen Oil Field, Gansu, and could degrade petroleum hydrocarbons efficiently at low temperature, will be selected as the study object. The complete genome information will be obtained using whole genome sequencing technology. The potential alkanes oxidase genes, gene cluster structure and transcriptional regulation factors will be analysed by bioinformatics. The function and metabolism mechanisms of alkanes oxidase genes will be illustrated by gene mutation technology combined with metabonomics technology. Furthermore, the potential novel transcriptional regulation factors will be screened using DNA affinity purification technology. The interaction between promoter sequences of alkanes oxidase genes and transcriptional regulation factors will be verified by gel retardation assay. Based on these results, the transcriptional regulation factors genes deletion mutation strains will be constructed according to the intention to explain detailedly the regulatory mechanisms of the petroleum hydrocarbons degradation. Therefore, new research thoughts focused on full developments of microbial functional genes, explanation of microbial degradation mechanisms of petroleum hydrocarbons, and design of genetic engineering strains with high metabolic activities will be likely come true according to the abovementioned results. As a consequence, Rhodococcus erythropolis KB1 has very important theoretical research and practical application values.