猪苓（Polyporus umbellatus）是我国传统药用真菌，其菌核富含多种生物活性成分，具有重要的药用价值。然而，猪苓菌核的形成机制复杂，受多种环境因素和生物因素的调控。课题组前期研究发现，草酸对猪苓菌核呈现典型的“低促高抑”的双相效应。在猪苓栽培生产中，蜜环菌（Armillaria spp.）是不可或缺的关键生物因子。本论文首先以营养琼脂培养基培养的猪苓菌丝/菌核为研究对象，为获取草酸影响猪苓菌核形成的差异蛋白谱与代谢谱，综合运用形态学、蛋白质组学、代谢组学以及化学成分分析等技术手段，系统探究了不同浓度外源性草酸对猪苓菌核发育的影响；其次，通过野外栽培试验，本论文探究了两种蜜环菌（A. gallica和A. mellea）影响猪苓菌核产量以及多糖与甾体类成分积累的规律，主要研究结果如下：

1. 不同浓度外源性草酸影响猪苓菌核发育的形态学及差异表达蛋白分析

通过石蜡切片和扫描电镜（SEM）观察发现，低浓度草酸（0.05 mg/mL）能够有效地促进猪苓菌核的生长发育，在相同时间内发育得更成熟，且菌核皮层细胞壁明显增厚，木质化程度提高，形成更致密的结构。基于4D-Label Free蛋白质组学技术检测了不同浓度外源性草酸影响猪苓菌核发育的差异蛋白表达谱，共鉴定到2902个蛋白，分析了与氧化应激、信号转导与细胞壁重建、防御相关以及多糖与甾体类成分合成相关的差异蛋白。利用ELISA技术测定Actin在不同浓度草酸影响猪苓菌核形成的差异表达水平，同时克隆了编码激酶结构域蛋白 PUPK和PUCE4的全长基因，以qRT-PCR技术进一步验证了它们在不同浓度草酸所培养的猪苓菌核/菌丝中的差异表达。

2. 非靶向代谢组学解析草酸影响猪苓菌核/菌丝药效学成分积累的规律

基于UPLC-MS技术的非靶向代谢组学分析表明，代谢物包括糖类、脂肪酸、氨基酸以及甾体类成分等。与对照组菌丝相比，低浓度草酸组菌丝与菌核中糖类成分、麦角甾醇与猪苓酮等成分含量高，而高浓度草酸组（1.00 mg/mL）中的药效学成分含量低。利用HPLC和苯酚-硫酸法测定不同草酸处理的菌丝和菌核的猪苓酮A、麦角甾醇和多糖含量，研究发现，低浓度外源性草酸组能促进猪苓多糖和甾体类成分增加，高浓度草酸组抑制猪苓活性成分的积累。

3. 蜜环菌侵染对猪苓菌核不同部位药效学成分积累的规律

在实验室无菌条件下培养的猪苓菌核虽有利于其发育机制的研究，但目前还无法实现将其推广应用至野外栽培试验。因此，我们以不同种类蜜环菌侵染猪苓菌核不同部位为切入点，探索其对猪苓药效成分积累的影响。基于前期从野生猪苓菌核分离获得不同种类的蜜环菌，通过比较Armillaria gallic和A. mellea侵染猪苓菌核后对其不同部位的甾体类成分与多糖含量检测发现，不同种类蜜环菌侵染的猪苓菌核随栽培年份的增长，猪苓中麦角甾醇、猪苓酮A、猪苓酮B和多糖等4种成分的含量及菌核生物量逐渐积累，猪苓隔离腔壁部位的4种成分显著高于其非侵染部位。与对照组A. mellea相比，A. gallica在促进猪苓产量与药效学成分积累方面具有明显优势。转录组数据分析显示，蜜环菌的侵染激活了猪苓菌核中多糖合成基因簇的表达，为猪苓菌核多糖合成提供了分子生物学证据支持。

本论文为不同浓度草酸影响猪苓发育机制的研究提供了重要的分子生物学证据，同时为阐明不同种类蜜环菌影响猪苓菌核化学成分积累的规律奠定了基础。

Polyporus umbellatus, a traditional medicinal fungus in China, produces sclerotia that are rich in various bioactive compounds with significant medicinal value. However, the mechanisms underlying sclerotial formation are complex and regulated by multiple environmental and biological factors. Previous studies by our research group have shown that oxalic acid exerts a typical biphasic effect on sclerotial development in P. umbellatus, characterized by stimulation at low concentrations and inhibition at high concentrations. In the cultivation of P. umbellatus, Armillaria spp. are indispensable symbiotic partners.

This study first used mycelia and sclerotia of P. umbellatus cultured on nutrient agar medium to explore the differential proteomic and metabolomic profiles associated with the effects of oxalic acid on sclerotial development. A combination of morphological observation, proteomics, metabolomics, and chemical component analysis was applied to systematically investigate the effects of exogenous oxalic acid at varying concentrations. Additionally, field cultivation experiments were conducted to study the impact of two Armillaria species (A. gallica and A. mellea) on the yield of sclerotia and the accumulation of polysaccharides and steroidal compounds. The main findings are as follows: 

1.   Morphological and Proteomic Analysis of Sclerotial Development Under Different Oxalic Acid Concentrations

Paraffin sectioning and scanning electron microscopy (SEM) revealed that low-concentration oxalic acid (0.05 mg/mL) significantly promoted sclerotial development, leading to earlier maturation, thickening of cortical cell walls, increased lignification, and formation of denser structures. A 4D-label free proteomic analysis identified 2,902 proteins differentially expressed under different oxalic acid concentrations. These included proteins related to oxidative stress, signal transduction, cell wall remodeling, defense response, and the biosynthesis of polysaccharides and steroidal compounds. ELISA assays were used to quantify the expression of Actin under various oxalic acid treatments. In addition, the full-length genes encoding the kinase domain proteins PUPK and PUCE4 were cloned, and their differential expression in mycelia and sclerotia was validated using qRT-PCR.

      2.   Non-targeted Metabolomic Analysis of Pharmacologically Active Compounds Affected by Oxalic Acid

Non-targeted metabolomics using UPLC-MS revealed that the metabolites affected by oxalic acid included sugars, fatty acids, amino acids, and steroidal compounds. Compared to the control group, low-concentration oxalic acid treatment led to higher levels of sugars, ergosterol, and polyporusterone in both mycelia and sclerotia, whereas high-concentration oxalic acid (1.00 mg/mL) inhibited the accumulation of these active compounds. HPLC and the phenol-sulfuric acid method were used to quantify the levels of polyporusterone A, ergosterol, and polysaccharides, confirming that low-concentration oxalic acid promotes the accumulation of bioactive components, while high concentrations suppress them.

      3.    Effect of Armillaria Infection on the Accumulation of Bioactive Compounds in Different Sclerotial Regions

Although axenic culture of P. umbellatus in the laboratory facilitates studies on developmental mechanisms, it is not yet applicable to field cultivation. Thus, this study further examined how infections by different Armillaria species affect the accumulation of bioactive components in various sclerotial regions. Two Armillaria species previously isolated from wild P. umbellatus were compared. Infections by A. gallica and A. mellea both promoted the accumulation of ergosterol, polyporusterone A and B, and polysaccharides over multiple years of cultivation. Notably, the compartment walls of the sclerotia showed significantly higher levels of these compounds than the non-infected regions. Compared to A. mellea, A. gallica exhibited a stronger ability to enhance both sclerotial yield and accumulation of pharmacologically active compounds. Transcriptomic analysis further showed that infection by Armillaria activated expression of gene clusters involved in polysaccharide biosynthesis in the sclerotia, providing molecular evidence for enhanced polysaccharide production.

This dissertation provides crucial molecular evidence for the effects of oxalic acid on the developmental mechanisms of P. umbellatus, and lays a foundational understanding of how different Armillaria species influence the accumulation of bioactive compounds in its sclerotia.