根癌农杆菌对苯酚的代谢途径及其调控机制的研究

Phenolics are important inducers of transgene process in Agrobacterium tumefaciens through its symbiosis with plant cells. Besides, A. tumefaciens can utilize some phenolic pollutants to independently and widely survive in soil. It is possible to achieve the regulation of transgenic process and the augment of its biodegradability by changing phenolic metabolic capacity of A. tumefaciens. There is still lack of comprehensive and mechanical knowledge of phenol metabolism in A. tumefaciens. In this project, phenol as the simplest phenolics is chosen as the research model. Combined with genome-scale modelling, transcriptome analysis, biochemical detection and molecular biology techniques, phenol metabolic process and related regulatory mechanism in A. tumefaciens will be studied: (1) Based on genome sequences and genome annotation, genome-scale metabolic model will be reconstructed using an integrated toolbox. (2) Phenol metabolic pathways will be predicted by genome-scale modelling and validated by the detection of gene expression and enzymatic activity，and gene knockout experiments. (3) Transcriptional regulatory modes of phenol metabolism will be analyzed from transcriptome data and confirmed by bacterial one-hybrid system, electrophoretic mobility shift assays, and DNase I foot-printing. The project will provide theoretical guide for other complex phenolic metabolism in A. tumefaciens, and enlighten to explore multifunction (such as biodegradation, transgene, and others) of microorganisms in nature.

酚类物质是根癌农杆菌与植物共生时转基因作用的重要诱导物；根癌农杆菌在土壤中独立生存时能够耐受和利用一些酚类污染物。通过改变根癌农杆菌对酚类的代谢能力，可能实现调控转基因过程和强化生物降解能力。然而，根癌农杆菌如何代谢和调控酚类降解，尚缺乏全局性和机理性的认识。本项目以苯酚这种最简单酚类为研究模型，借助基因组规模模拟和转录组分析，结合生化检测和分子生物学手段，对根癌农杆菌的苯酚代谢过程和调控机制进行研究：(1)基于根癌农杆菌基因组序列和序列注释，采用集成工具箱构建基因组规模代谢模型；(2)基于模型模拟根癌农杆菌的苯酚代谢过程，通过检测基因表达和酶活性、基因敲除与表达等策略验证途径；(3)基于转录组数据预测苯酚代谢的转录调控模式，利用细菌单杂交、凝胶迁移、DNaseI足迹实验验证。研究结果将为根癌农杆菌代谢其他酚类提供参考，对于发掘自然界中具有生物降解、转基因功能等多功能微生物有指导意义。

根癌农杆菌既可以在土壤中独立生存，也可以与植物共生、侵染植物而被广泛用作植物转基因工具。酚类物质是根癌农杆菌的转基因过程重要的诱导物之一。在自然状态下，根癌农杆菌要实现对宿主侵染，需要识别伤口分泌的酚类等营养物质，趋化作用是第一步。根癌农杆菌从独立生存转向侵染植物，是根癌农杆菌环境适应性行为过程，受动态代谢调控。为了解析酚类对根癌农杆菌的调控机制，本项目对根癌农杆菌趋化和代谢途径进行了全面、系统的研究，并且借助计算模拟，结合生化检测和分子生物学手段，初步筛选得到Atu0387参与到根癌农杆菌对酚类物质的响应。研究结果为系统性的认知和解析根癌农杆菌和植物相互作用，进一步揭示根癌农杆菌复杂的致病机制奠定了一些研究基础。