手参 Gymnadenia conopsea （L.）R.Br. 是一种广域零星分布的地生兰科植物， 具有填精补髓、增强体质等功效，是我国的传统中药，也是蒙药、藏药常用药。 手参现为我国二级保护植物，其种子微小，无胚乳，自然条件下多为无性繁殖， 需要依赖 共生真 菌 为其提供 营养 才 能 萌发。前 期研究 发 现角担菌 属真菌Ceratobasidium GS2 能够促进手参种子萌发。本文以 Ceratobasidium GS2、手参种子和手参种子共生萌发形成的原球茎为主要研究对象，对其产生的次级代谢产物 进行研究，运用硅胶柱色谱、凝胶色谱、半制备液相色谱等技术进行化合物的分 离纯化，结合核磁共振、高分辨质谱等多种分析鉴定方法进行化合物的结构鉴定， 并验证部分化合物的生物活性；同时采用代谢组学技术对手参种子真菌共生萌发 过程进行了研究，为全面解析真菌促进手参种子萌发的物质基础提供支撑。 本研究共得到 23 个化合物，从共生萌发真菌 Ceratobasidium GS2 中分离得到 15 个化合物（1-15），包括 7 个甾类化合物（1-7）、6 个呋喃类化合物（8-13）以 及 2 个简单化合物（14-15），其中化合物 8 为新化合物，均首次从角担菌属真菌中 分离得到。通过 Mosher 反应确定化合物 8 的立体化学结构，发现化合物 8 的羟基 在 Mosher 试剂中不稳定，会产生 4 个类似物 16-19。通过 UPLC-Q-TOF-MS/MS 对 这些降解产物进行了鉴定，分离纯化出化合物 18，并通过核磁共振对其结构进行 了表征。最终通过计算 ECD 确定了化合物 8 的绝对构型。对部分单体化合物进行 了手参种子萌发活性实验，发现甾类化合物（1-6）可以促进手参原球茎的生长发 育，呋喃类化合物（10-11）对 Ceratobasidium GS2 的生长以及手参种子萌发表现 出较强的抑制活性。同时化合物 8-12 和 18 具有较弱的抗氧化活性，化合物 9-11 对狗尾草（Setaria viridis）具有植物毒性。 从手参种子乙腈提取物中分离得到 6 个化合物（20-25），包括联苄类、聚酮类、 吲哚类、甾醇类以及苯环类化合物。化合物的结构通过波谱分析技术和文献比对 确定。手参种子中化合物 22 和 25 与 Ceratobasidium GS2 的化学成分相同。联苄类 化合物在手参块茎化学成分中已有报道。 从手参种子共生萌发形成的原球茎中分离得到 2 个化合物（26-27），通过一维 核磁共振技术和文献比对确定为糖苷类和苯环类化合物。这 2 个化合物都曾在手参 块茎以及其他兰科植物中被分离得到。 利用 UPLC-Q-TOF-MS/MS 技术对比分析了 Ceratobasidium GS2 菌丝在纯培养 和与种子共生条件下的代谢变化，发现代谢物的积累在两组之间存在显著差异。 共注释到 1281 个代谢物，筛选出 958 个差异代谢物，其中 514 个上调表达，444 个 下调表达。共生组的 KEGG 功能注释结果表明差异显著富集的是氨基酸生物合成 途径，显著富集的通路主要包括精氨酸生物合成、赖氨酸生物合成等途径，这暗 IV 示在共生过程中可能发生氨基酸的营养交换。在共培养 Ceratobasidium GS2 菌丝中 检测到 26 个纯培养菌丝中没有的化合物，主要包括糖类、氨基酸等初级代谢产物 和萜类、黄酮类等次级代谢产物。本文的研究结果将为全面解析真菌促进手参种 子萌发的机制奠定基础。

Terrestrial orchid Gymnadenia conopsea is widely used for Tibetan medicine and Mongolian medicine, having evident pharmacological activities. Due to their tiny seeds devoid of endosperm, most orchid species are dependent on mycorrhizal fungi to supply the necessary nutrients for seed germination during early stages of plant development as well as in some adult orchid species. The fungal strain Ceratobasidium GS2, initially isolated from roots of G. conopsea, was able to support seed germination under both artificial and natural conditions. In this research, secondary metabolites of Ceratobasidium GS2, seeds and protocorm of G. conopsea were investigated using various chromatographic columns and their structures were identified by multiple spectroscopic methods. As a result, 23 compounds including 1 new compound were isolated. 15 compounds (1-15) including seven sterols (1-7) and five furanoids (8-13) were isolated from the symbiotic fungus Ceratobasidium sp. GS2. 6 known compounds (20-25) from seeds of G. conopsea and 2 known compounds (26-27) from protocorm of G. conopsea were identified. This was the first chemical investigation of the symbiotic fungus, seeds and protocorm of G. conopsea. The structure of new compound 8 was determined mainly based on HR-ESI-MS and NMR spectral data. Modified Mosher’s reactions were used to establish the stereochemistry of the hydroxyl group in 8, which was not stable in Mosher’s reagents and transformed into four analogs 16-19. These degraded products (16-19) were elucidated based on UPLC-Q-TOF-MS/MS analysis, and compound 18 was further isolated from the degraded mixture and its structure was characterized through NMR experiments. Therefore, the absolute configuration of compound 8 was determined by ECD combined with quantum-chemical calculations adopting time-dependent density functional theory (TDDFT). Compounds 1-6 can promote the protocorm of G. conopsea formation and development, but compounds 10-11 strongly showed inhibition on the seed germination. In addition, compounds 8-12, and 18 showed weak antioxidant activities, and compounds 9-11 displayed phytotoxicity on punctured detached green foxtail leaves. UPLC-Q-TOF-MS/MS technology was used to explore the effect of metabolites of Ceratobasidium GS2 on symbiosis with seeds. A total of 1281 metabolites were annotated and 958 differential metabolites were screened, including 514 up-regulated metabolites and 444 down-regulated metabolites. The results of KEGG function annotation in the Ceratobasidium GS2 symbiotic group showed that the differentially expressed metabolites VI were significantly enriched in biosynthesis of amino acids. 26 compounds not found in the blank group were detected. The results will further lay a good foundation for symbiosis germination.