甲基丁香酚在千层金叶片精油抑制紫色杆菌群体感应中的作用机制研究

The leaves of Melaleuca bracteata are rich in essential oil which emit pleasant aroma and have the activity of quorum sensing inhibition in gram-negative bacteria. In our previous study, an active compound, methyleugenol identified by GC-MS and 2D-NMR, with high QS inhibition activity was purified from essential oil of Melaleuca bracteata leaves, which is expected to be developed as a novel biological anti-bactericide targeting QS of plant pathogenic bacteria. However, the molecular mechanism of inhibition in bacterial QS effect by methyleugenol is still not clear at present. In this project, methyleugenol of high purity will be used to treat Chromobacterium violaceum ATCC12472 to determine its effects on QS signaling and physiology. The production of signal molecule C10-HSL will be evaluated by TLC combining with GC-MS. qRT-PCR will be performed to investigate the the gene expression of cviR，cviI，vioB，pslA，lasA and lasB. The binding sites of methyleugenol onto signal receptor CviR and signal molecule synthetase CviI will be studied using molecular docking analysis. The quantitative production of violacein and biofilm will be tested, and the formation and structure changes of biofilm will be analyzed under light and confocal laser scanning microscopy. The project aims at elucidating the mechanism of quorum sensing inhibition of gram-negative bacteria by methyleugenol from essential oil of Melaleuca bracteata leaves, which will provide the theoretical basis and working foundation for further studies on the mechanism of quorum quenching and pathogenicity inhibition and the development of novel biological anti-bactericide agents.

千层金叶片富含精油，芳香怡人，具有抑制细菌群体感应（Quorum Sensing，QS）作用。课题组前期从千层金叶片精油中纯化到一个高QS抑制活性的化合物，经鉴定为甲基丁香酚，有望开发成以植物病原细菌QS系统为靶标的新型生物农药，但其抑制细菌QS作用的机制仍不清楚。本项目拟以此为基础，用甲基丁香酚处理紫色杆菌，通过TLC结合GC-MS分析紫色杆菌信号分子C10-HSL变化；荧光定量PCR检测QS系统相关基因cviR、cviI、vioB、pslA、lasA和lasB转录水平；分子对接分析甲基丁香酚和分子受体蛋白CviR、信号分子合成酶CviI形成复合物的结合位点；定量检测紫色菌素和生物膜，显微镜观察生物膜形成和结构变化。旨在明确甲基丁香酚在千层金叶片精油抑制革兰氏阴性细菌QS中的作用机制，为进一步研究甲基丁香酚抑制植物病原细菌QS与致病性的机制提供理论依据和工作基础，指导千层金精油生产开发。

群体感应 (quorum sensing)是细菌根据细胞密度变化进行基因表达调控的一种生理行为。具有群体感应的细菌能产生并释放一种被称为自体诱导物(autoinducer)的信号分子，它随着细胞密度增加而同步增加.当自体诱导物积累到一定浓度时会改变细菌特定基因的表达。植物中的芳香化合物能够抑制细菌的群体感应。本课题以千层金精油的主要成分甲基丁香酚为主要研究对象，从生理生化和分子层面，研究了甲基丁香酚对紫色杆菌群体感应的影响并对其抑制机制进行了探讨: (1)甲基丁香酚（ME）能够抑制紫色杆菌色素的产生、生物膜形成及群集运动。(2) 甲基丁香酚能从分子水平上抑制QS相关基因的转录表达。（3）甲基丁香酚抑制紫色杆菌群体感应可能通过CviR蛋白互作和降解信号分子（C6-HSL）来实现，从而减弱细菌致病性。因此甲基丁香酚可作为潜在的细菌群体感应抑制剂。