假单胞菌JY-Q中尼古丁代谢同工簇功能和互作机制研究

重复基因在真核生物中较常见。近年来也有细菌发现重复同源基因（簇）的少量报道。从高浓度废烟叶水提液中分离的假单胞菌JY-Q显示耐受和降解高浓度尼古丁能力。比较基因组学分析发现JY-Q尼古丁代谢途径的上，中，下游功能模块有两个以上同源基因簇（同工簇）组成。且发现同工簇发挥了促进作用，帮助其在高浓度尼古丁环境中生存和生长，但各同工簇的贡献差异显著。本研究拟通过基因敲除、荧光定量PCR和酶反应动力学分析，考察在有无尼古丁条件下，JY-Q各同工簇内基因和同工簇间基因的功能差异及其互作关系。通过同功能模块内同工簇的启动子互换和优化，及同工簇潜在调控因子挖掘和突变，探明同工簇差异调控及其互作机制，进而探究同步强化同功簇表达这种新策略对提高菌株降解性能的效果。新分离芽孢杆菌ZutR对高浓度尼古丁的耐受可能也是重复基因簇造成的，其基因组和差异表达研究，将辅助探明同工簇在细菌适应高浓度尼古丁中的作用机制。

Duplicated genes (clusters) commonly exist in eukaryotes. However, in past decade, a few publications reported that there also existed duplicated genes (clusters) in prokaryotes. Pseudomonas sp. JY-Q and Bacillus sp. ZutR isolated from the nicotine-rich water extract of tobacco waste showed the ability to tolerate and degrade high concentrations of nicotine. Comparative genomics analysis revealed that there were more than two homologous gene clusters (isoclusters) in the upper, middle, and lower functional modules of nicotine metabolism pathway in the JY-Q genome. Previous studies have found these coexisting homologous/isotropic clusters play a promoting role and help them survive and grow in high nicotine concentrations, but the contributions of isotropic clusters are different. In this study, gene knockout, fluorescence quantitative PCR and enzyme reaction kinetics were used to investigate the functional differences and interactions of genes within and among isoclusters under different nutritional conditions (nicotine or glucose). Furthermore, the promoter interchange and optimization of the same functional module, as well as the mining and mutation of the potential regulatory factors of the same functional module, will be used to reveal the different regulatory mechanism of the same functional module. Meanwhile, based on the comparative genomics analysis of strain ZutR, the function of duplicated gene cluster in bacteria on the adaption ability and degradation activity of environment pollutants will be revealed. Finally, the expression of isocluster could be enhanced synchronously by promoter optimization in order to further improve the nicotine degradation function of the strain, which strategy would be potential for other bacteria capable degrading pullutants.