萘降解菌Pseudomonas putida ND6中接合转移系统的作用机制及环境迁移性能研究

Conjugative transfer is the most important means of improving evolution and spreading biodegrading ability among bacteria. In Pseudomonas putida ND6, the naphthalene-degrading plasmid pND6-1 could translocate across the cell envelopes of two cells with the mediation of the conjugation systems encoded by another plasmid. To date, little research was conducted on the comprehensive biochemical and genetic examination of the conjugative transfer of naphthalene-degrading plasmid with the help of other plasmids, its assembling model and mechanism had not been elucidated. In this study, we focus on the two aspects of conjugation in strain ND6. First is to study on the mechanism of the conjugative transfer system in P. putida ND6. Based on the technology of membrane protein separation, construction of recombinant strain and quantative proteomics, we will determine the localization and interaction of proteins involved in conjugation system, demonstrate the assembling model and mechanism of conjugation in strain ND6. Second, monitoring techniques will be designed to quantitatively follow conjugative plasmid transfer under different conditions and in distinct environments. Different genes encoding fluorescence proteins and antibiotics will be inserted into the genome and plasmids, respectively. In this way, the fate of plasmids in a bacterial community can be monitored in situ nondestructively, and the library of the recipient bacteria for conjugation will be constructed.

接合转移对于细菌的进化及降解基因在环境中的扩散具有重要意义。研究表明Pseudomonas putida ND6中的萘降解质粒pND6-1具有接合转移的能力，但这种能力是由另一质粒pND6-2编码的接合转移系统介导的。目前国内外还没有一质粒介导另一萘降解质粒接合转移的相关报道，其装配模式和作用机制还不是十分明了。因此，本研究计划开展两部分工作。第一，萘降解菌ND6中接合转移系统的作用机制研究。主要通过膜蛋白分离、基因敲除、定量蛋白质组学等技术手段，确定pND6-2质粒上接合转移相关蛋白的定位，明确蛋白之间的相互作用，从而阐明其接合转移系统的装配模型和作用机制。第二，P. putida ND6中萘降解基因在环境中的迁移效能研究。主要通过荧光标记、抗性基因标记等方法，实时监测萘降解质粒pND6-1在环境中的迁移，鉴定接受萘降解基因的受体菌的种属，构建适用于ND6菌株的接合转移受体菌库。

putida ND6中的萘降解质粒pND6-1具有接合转移的能力，但这种能力是由另一质粒pND6-2编码的接合转移系统介导的。目前国内外还没有一质粒介导另一萘降解质粒接合转移的相关报道，其装配模式和作用机制还不是十分明了。因此，本研究共开展了两部分工作。第一，萘降解菌ND6中接合转移系统的作用机制研究。主要通过基因敲除、定量PCR学等技术手段，研究了pND6-2质粒上接合转移系统中核心复合体相关蛋白，及其对接合转移的影响。第二，P. putida ND6中了质粒pND6-1和pND6-2在环境中的迁移效能研究。主要通过抗性基因标记等方法，研究了不同因素对两个质粒接合转移的影响及对萘降解的意义，监测两个质粒在环境中，尤其是水环境和活性污泥中的迁移性能。