石蒜内生真菌LrLF6促进宿主加兰他敏积累的机制研究

Galanthamine, which specifically exist in Amaryllidaceae plants, is used clinically for treating Alzheimer. At present, galanthamine is mainly extracted from Lycoris radiata. Previously, fungal endophytes in L. radiata have been isolated and identified in our lab. By inoculating fungal endophytes to L. radiata aseptic tissue culture plantlets, we get a fungal endophyte LrLF6 that can significantly increase the galanthamine accumulation in L. radiata. However, the mechanisms underlying fungal endophyte–induced galanthamine accumulation in L. radiata are obscure. In this application, the transcriptomes of fungal endophyte–colonized and control plantlets will be constructed and compared. The expression changes of genes related to primary metabolism, defense responses and secondary metabolism in L. radiata arose from fungal endophyte LrLF6 colonization will be revealed. Moreover, effects of fungal endophyte on plant photosynthesis, root development and nitrogen uptake will be detected, which can be used to analyze whether primary metabolism provides materials and energy for galanthamine synthesis. Further, on the basis of the information obtained from transcriptomes, plant defense signaling cascades activated by fungal endophyte will be deciphered using liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS), which will be further used to elucidate the effects of various signaling molecules on the galanthamine synthesis–related gene expressions. In brief, the results obtained here will dissect the mechanisms of fungal endophyte–induced galanthamine accumulation in L. radiata from two aspects: the one is that fungal endophyte can enhance the plant primary metabolism; the other is that fungal endophyte can activate the plant defense responses. This application will provide the theoretical direction to utilize fungal endophytes to promote galanthamine accumulation in L. radiata. Furthermore, this study will prefect the interaction mechanisms between plants and fungal endophytes.

加兰他敏是石蒜科植物特有的生物碱，被广泛应用于阿尔兹海默症（早老性老年痴呆症）的临床治疗，目前其主要从石蒜中提取。前期，申请人从石蒜体内分离、筛选到一株能够显著促进宿主中加兰他敏积累的内生真菌LrLF6。为了进一步分析LrLF6促进石蒜体内加兰他敏积累的机制，本申请项目拟通过比较转录组测序，初步揭示内生真菌对石蒜初级代谢、防御响应、次级代谢的影响；进一步检测内生真菌对石蒜光合作用、根系发育、氮素吸收等的改变，分析初级代谢是否为加兰他敏合成提供了物质和能量基础；结合比较转录组数据，使用液质联用、气质联用等技术，明确内生真菌激活的植物防御信号级联，分析各信号分子对加兰他敏合成关键基因表达的影响。以上研究将从促进植物初级代谢和诱导植物防御响应这两个方面揭示内生真菌促进石蒜加兰他敏积累的机制，为更加高效地利用内生真菌促进加兰他敏的积累提供理论指导，也将丰富植物和内生真菌的互作机制。

石蒜科生物碱特异地存在于石蒜科植物中，具多种药用活性，目前主要从植物中提取获得。然而，生物碱在植物体内含量很低，如何有效促进生物碱的积累是药材栽培中亟需解决的问题。石蒜（Lycoris radiata）是石蒜科生物碱的主要药源植物，因此，本项目以石蒜为研究对象，分析内生菌对生物碱积累的影响。.首先，建立了5种主要生物碱（水仙环素、石蒜碱、加兰他敏、力可拉敏、多花水仙碱）的检测方法。之后，通过ITS扩增子测序发现，高生物碱含量的石蒜和低生物碱含量的江苏石蒜（Lycoris houdyshelii）中内生真菌群落组成差异显著，生物碱含量与内生真菌丰度相关。同时，分离了石蒜花、花葶、叶、鳞茎、根中的内生真菌，共106株，属于27个种，经相关性分析和两次独立盆栽实验，得到9株显著促进石蒜中生物碱积累的菌株。进一步，建立了内生真菌与石蒜无菌组培苗一对一的互作体系，发现6株内生真菌能显著促进至少一个组织中一种生物碱的积累；此外，Cylindrocarpon olidum LrBF4和Galactomyces pseudocandidum LrRF9显著增加了石蒜中茉莉酸（JA）信号途径相关基因的表达，表明JA信号参与了内生真菌促进石蒜科生物碱的积累。.同时，分析了石蒜各组织内生细菌的群落组成和多样性，分离到188株可继代培养的内生细菌，属于30个属；其中多株具有吲哚乙酸合成、固氮、铁载体合成等能力；盆栽实验表明，3株内生细菌显著增加石蒜的生物量，14株显著促进石蒜不同组织中至少一种生物碱的积累。16S rDNA扩增子测序显示多种生物碱影响内生细菌的群落组成和分布且多个细菌类群与生物碱含量呈显著负相关；利用分类学地位相同的可培养内生细菌菌株和生物碱标准品进行验证，发现石蒜科生物碱能抑制特定类群内生细菌的生长，表明内生细菌定殖石蒜后，诱导生物碱积累增加，而具抑菌活性的生物碱能调节内生细菌的生长和分布，最后，达到“拮抗平衡”，维持和谐共生。.综上，本项目研究成果为实现石蒜栽培中高品质药材的获得和特定药用成分的诱导提供了理论依据和技术支持，内生菌菌剂的开发和利用在药材栽培中具有极大的应用潜力。