假单胞菌LY1降解3-吲哚乙酸上游途径分子机理研究

Indole-3-acetic acid, an important indole derivative, is a central phytohormone, which regulates the whole repertoire of plant developmental processes. The balance of indole-3-acetic acid concentration is important for the health of plant, and excess indole-3-acetic acid produced by rhizosphere microorganisms is harmful to the plant and other microorganisms. Indole-3-acetic acid degradation microorganisms can regulate the concentration of indole-3-acetic acid in soil, and decrease the inhibition caused by high concentration of indole-3-acetic acid. Therefore, the microbial degradation of indole-3-acetic acid is important for the health of soil. However, in the past years, the study of indole-3-acetic acid degradation process by microorganisms has been neglected for a long time..In this study, the upper pathway of idole-3-acetic acid degradation in the strain Pseudomonas sp. LY1 will be characterized. Based on the genome and transcriptome information of strain LY1, the key genes in indole-3-acetic acid degradation will be identified by gene knockout and gene complement. The essential genes in indole-3-acetic acid degradation will be cloned and expressed in E. coli, and the proteins expressed by the genes will be purified. The basic biochemical properties of the enzymes will be characterized, and the catalyzing mechanism of these enzymes will be investigated by isotope labeling, Stopped-Flow and circular dichroism spectrum experiments. The results of this study will lay a solid foundation for the in-depth study of the mechanism and physiological of microbial degradation of indole and its derivatives.

吲哚乙酸是一种重要的吲哚衍生物，适宜浓度的吲哚乙酸是土壤保持活力的重要因素。过量的吲哚乙酸会对植物和微生物造成危害，吲哚乙酸降解微生物能够调节土壤中吲哚乙酸的浓度，解除过量吲哚乙酸造成的危害，对维持土壤健康起到了重要作用。研究吲哚乙酸的微生物降解十分必要，然而该方向的研究长期被忽视。申请人团队分离并鉴定了一株吲哚乙酸降解菌株Pseudomonas sp. LY1，本项目在该菌株现有工作基础上拟进行以下研究：以基因组、转录组数据为基础，利用基因敲除和回补定位功能基因，对关键基因进行体外克隆表达，蛋白纯化；研究酶的基本生化性质，利用Stopped-Flow、圆二色谱等技术研究关键酶的催化机理，揭示菌株LY1吲哚乙酸上游降解途径的分子机理。本研究将为微生物代谢吲哚类化合物的分子机理研究提供参考。

吲哚乙酸是一种重要的植物生长素，广泛的分布于植物体内和根际区域。在植物生长，植物与微生物互作中扮演了重要的角色。吲哚乙酸的微生物降解对于微生物植物互作及维持根际吲哚乙酸动态平衡起到了重要作用，但是微生物降解吲哚乙酸的分子机制仍不清楚。本项目分离并鉴定了一株能够以吲哚乙酸作为唯一碳、氮源进行生长的菌株Pseudomonas sp. LY1，本项目利用HPLC、LC-MS等技术对中间代谢产物进行了分离、纯化和鉴定，并解析了吲哚乙酸的降解途径。吲哚乙酸首先生成dioxindole-3-acetic acid，随后可以通过2条途径进行后续降解，第一条途径中dioxindole-3-acetic acid被进一步降解为靛红，靛红被催化或自我分解为靛红酸，靛红酸会被转化为邻氨基苯甲酸。另一条途径中dioxindole-3-acetic acid会生成邻苯二酚，并通原儿茶酸途径降解。这是首次在一个菌株中同时发现吲哚化合物降解的两条主要代谢途径。进一步，基于基因组、蛋白质组和生物信息学分析，本研究发现了一个与吲哚乙酸降解的基因簇iad cluster，该基因簇的表达受到了吲哚乙酸的诱导，蛋白质组数据也证明在吲哚乙酸存在的情况下，该基因簇上面的蛋白表达量显著上升（1.5~100倍）。在该基因簇上面发现了与吲哚乙酸降解相关的基因簇iadHABICDEFG，与邻苯二酚和原儿茶酸降解的基因簇catBCA和pcaABCDEF。通过基因敲除和回补，证明了iadA负责催化吲哚乙酸降解的第一步，吲哚乙酸转化为dioxindole-3-acetic acid的反应，而iadB负责催化dioxindole-3-acetic acid的转化。本项目首次利用蛋白质组分析进行了微生物降解吲哚类化合物的分析研究，并且发现了IadB催化了dioxindole-3-acetic acid的降解。本研究将为后续吲哚类化合物的降解提供参考。