除草剂利谷隆的微生物代谢机制与矿化关键菌群的结构与功能

Linuron is one of the most widely used phenylurea herbicides in the world. Linuron and its metabolites are frequently detected as contaminants in ecosystems. The environmental fate of linuron, including its microbial degradation, has caused great concern. However, the catalytic mechanism of amidohydrolase with broad substrate range in Gram negative strain and the transport mechanism of linuron remains unclear. The relationships between the community structure of 3,4-dichloroanine-mineralizing microorganisms and the degrading efficiency of the enrichment have not been elucidated. In this grant, a stable enrichment capable of mineralizing linuron will be selected as the material for investigation. The amidohydrolase gene responsible for the breaking the amide bond of linuron to produce 3,4-dichloroanine will be cloned from the Gram negative strain of Diaphorobacter sp. LR2014-1, which is capable of degrading broad range of phenylurea herbicides and the catalytic mechanism of the amidohydrolase will be elucidated. The transport for linuron will be identified and the mechanism for linuron transportation will also be clarified. In the community level, the key consortia involved in the mineralizing 3,4-dichloroanine in the enrichment will be elucidated based on the technology of DNA based stable isotope probing, and the relationships between the community structure of 3,4-dichloroanine-mineralizing microorganisms, the degrading efficiency of the enrichment as well as the environmental factors of the enrichment will also be investigated. The progress of this grant will enhance our insights into the microbial catabolism of linuron and provide technological support for the biodegradation of linuron, and even provide a promising candidate for construction of phenylurea herbicide-resistant genetically modified crops and trans-genetic plants for bioremediation of linuron-contaminated sites.

利谷隆是全世界使用最广泛的取代脲类除草剂之一，利谷隆及其中间代谢产物3,4-二氯苯胺造成的环境污染受到广泛关注，微生物降解是环境中利谷隆及其产物消除的主要方式。然而，关于阴性细菌中利谷隆广谱酰胺水解酶的催化机制、矿化菌群结构与功能之间的关联还没得到有效阐明。本项目以一利谷隆矿化富集液为研究对象，从富集液中分离的对取代脲类除草剂具有广谱降解效果的菌株Diaphorobacter sp. LR2014-1中克隆断裂利谷隆脲桥产生3,4-二氯苯胺的酰胺水解酶基因，研究酰胺水解酶的催化机制；鉴定利谷隆的转运蛋白并揭示其转运过程；从群体层面研究参与3,4-二氯苯胺矿化的关键微生物菌群，阐明3,4-二氯苯胺的降解效率和微生物菌群结构、环境条件之间的内在关联，为利谷隆的微生物降解与修复提供理论指导与技术支持，甚至为抗除草剂转基因作物的培育和转基因植物修复除草剂污染环境提供优良基因资源。

利谷隆及其代谢产物3,4-二氯苯胺（3,4-DCA）均为优先控制污染物。微生物降解被认为是环境中利谷隆和3,4-DCA消除的主要方式。本项目通过前期获得一份可以完全降解利谷隆的富集液，高通量测序分析表明其群落组成以Methylobacillus属为主，其次为Flavobacteriaceae属，Pseudomonas属和Achromobacter属等，同时还存在丰度较低的Chitinophagaceae属和Diaphorobater属。利用稀释涂布法，从中分离到一株利谷隆高效降解菌株Diaphorobacter sp. LR2014-1和三株3,4-DCA的矿化菌株Achromobacter sp. S9-254a/Amp-2/ANB-1。菌株 LR2014-1不能矿化利谷隆，而是将其转化为等摩尔的3,4-DCA。通过基因组测序技术，从菌株LR2014-1中同时克隆到两个广谱的取代脲类除草剂水解酶基因 (phh & tccA2)。Phh为首次在革兰氏阴性细菌中发现的能同时降解N,N-二甲基和N-甲氧基-N-甲基取代脲类除草剂的水解酶。TccA2为首次报道的取代脲类除草剂环草隆水解酶。菌株S9-254a、Amp-2和ANB-1均能以3,4-DCA为唯一碳源生长，且菌株LR2014-1与菌株ANB-1可以协同矿化利谷隆。借助稳定性同位素核酸探针技术，最终将富集液中部分参与3,4-DCA降解的关键微生物菌群成功标记。利用高通量测序技术分析发现，非标记组和标记组在重层的微生物群落组成发生显著变化。在门水平上，标记组重层中的Proteobacteria门类群含量显著上升。在属水平上，标记组重层中的Diaphorobacter属，Achromobacter属和Staphylococcus属等丰度显著增加，暗示其在3,4-DCA的降解过程中起了重要作用。通过在不同因素条件下培养3,4-DCA矿化富集液，获得了具有不同降解效率的3,4-DCA矿化富集液。借助稳定性同位素探针技术，成功将部分参与3,4-DCA降解的功能微生物菌群标记上。通过高通量测序技术，阐明富集液降解3,4-DCA的效率与富集液中关键微生物菌群的结构之间的内在关系。