真菌激发子对桑黄三萜合成的诱导及作用机制研究

Sanghuangporus sanghuang is one of the most famous medicinal mushroom in China obligating parasitic on morus trees. It has become a hot spot of current research in the world according to its good pharmacological activities. Triterpenoid is a kind of important secondary metabolites of S. sanghuang with good pharmacological activities of anticancer and anti-aging, etc. The potential for development and utilization of this kind of resource was huge. In order to greatly improve the efficiency of liquid fermentation of S. sanghuang and explore the synthesis rule of triterpenoid, the methods of spectrum analysis and biological study were used together in this study. The research of fungal elicitor polysaccharides structure analysis and the separation, purification and identification of S. sanghuang triterpenoid were carried out. The effects of fungi elicitor with different structural composition on the dynamic change of S. sanghuang total triterpenoid, several monomer triterpene, lanosterol and ergosterol were detected. Correlation analysis was carried out on all the results above to discuss the possible structure-activity relationship between the structural composition of fungal elicitor, shch as polysaccharide composition, molecular weight distribution, monosaccharide composition, glycosidic bond type, sugar ring structure and the functional group kinds and number, and the biosynthetic of S. sanghuang triterpenoid. The possible pathway of S.sanghuang triterpenoid biosynthesis was explored simultaneous. All the results were used to reveal the action mechanism of the induction of fungal elicitor. This study will provide a new idea to S. sanghuang triterpenoid stimulation and a promising theoretical basis and technical support for the development of this kind of valuable medicinal mushroom.

桑黄是我国最为著名的药用真菌之一，专性寄生于桑属树木上，因具有良好的药理活性而成为目前国内外研究的热点。三萜类成分是桑黄中一类主要的次生代谢产物，具有抑制多种癌细胞生长以及抗衰老等活性，开发利用潜力巨大。为大幅提高桑黄液体发酵效率，探索其三萜生物合成规律，采用波谱分析与生物学研究相结合的方法，从真菌激发子的多糖结构组成分析及桑黄三萜类化合物的分离、纯化与鉴定研究入手，通过观测不同多糖结构组成的真菌激发子对桑黄总三萜、几种单体三萜及羊毛甾醇和麦角固醇等动态变化的影响并进行相关性分析，探讨真菌激发子的多糖分子量分布、单糖组成、糖苷键类型、糖环结构、官能团种类数量等结构特征与三萜类化合物生物合成之间的构效关系以及桑黄三萜生物合成途径，进一步揭示其诱导作用机制，为桑黄液体发酵三萜增产研究提供一条新思路，并为这一宝贵药用真菌资源的开发利用提供良好的理论基础和技术支撑。

桑黄是我国著名的珍稀药用真菌。三萜类成分是桑黄中主要的次生代谢产物，具有多种药理活性，开发利用潜力大。为大幅提高桑黄液体发酵效率，以筛选出的薄多年卧孔菌胞外多糖激发子（P激发子）在桑黄液体发酵过程中添加，开展了真菌激发子对桑黄三萜合成的诱导及作用机制研究。在培养的第4d，激发组(ET)菌球三萜合成途径中鲨烯合酶（SE）和羊毛甾醇合酶（LS）两种关键酶基因表达明显上调，NOS活性、NO含量显著高于未激发组（WET），在第5d，菌球中三萜类代谢前体物质羊毛甾醇含量达最高值，为WET组的45倍，胞内总三萜含量比WET组提高37.11%，激发效果显著。激发子通过诱导NO和NOS的信号传递，进而促进了胞内三萜的积累。构效关系研究的结果显示，P激发子所带氯代烷烃官能团可能存在积极的激发效果。. 次生代谢组学的分析结果显示，代谢产物在ET和WET组间存在显著差异。差异代谢物中共检测到3种三萜类化合物:白桦脂醇、白桦脂酸和2-羟基齐墩果酸。ET中的白桦酯酸是WET中的2.62倍；2-羟基齐墩果酸为WET的114.67倍，激发效果显著。桑黄菌球的全长转录组测序，共获得30,704转录本序列，筛选到ET与WET组间存在的97个差异转录本，其中57个上调。从差异代谢基因注释及富集分析的结果来看，P激发子可有效促进桑黄三萜及倍半萜合成途径的差异基因富集，并在桑黄次级代谢途径及防卫基因活化以及信号转导等方面发挥重要作用。结合代谢组和转录组对KEGG通路分析的结果发现，桑黄的萜类生物合成途径可能为甲羟戊酸途径。桑黄萜类代谢与赖氨酸丙二酰化修饰关联分析的结果发现，共有26种与三萜和多糖生物合成相关的酶被丙二酰化，大量的酶在糖酵解和TCA循环中得到丙二酰化的支持，说明丙二酰化可能与细胞内三萜、多糖等的合成途径密切相关。. 研究结果为桑黄液体发酵三萜增产研究提供了一条新思路，并为这一宝贵药用真菌资源的开发利用提供良好的理论基础和技术支撑。