基于真菌激发子调控的桑黄活性多糖分子链空间构象研究

Phellinus igniarius is one of the most well-known medicinal mushrooms in traditional Chinese medicine. The polysaccharide of Phellinus igniarius has been reported to present obvious immunomodulatory activity. The immunomodulatory activity of fungi polysaccharide could be stimulated by recognition of macrophages and its activity is supposed to be with the certain advanced structure, such as chain conformation, mechanical property and dynamics performance. Up to now, employment of pathogenic and non-pathogenic fungal preparations and chemicals, termed as elicitors, thus becomes one of the most important strategies to improve secondary metabolite production in cell cultures. Fungal elicitors were induced to cell culture process at the later-stage of exponential growth phase, which was proved to be an effective way to increase the production and productivity of secondary metabolites. However, no study has been reported on the synthetic mechanism of phellinus igniarius polysaccharide which is regulated by elicitors. Besides, there is few literature on the relationship between structure-activity and the active polysaccharide from Phellinus igniarius which is regulated by fungal elicitor. . In this project, we would analyse the activity ofα-PGM and PGI, studying the change of RNA expression by transcriptome. Then the effect of fungal elicitor on gene expression and protein expression during polysaccharide synthesis would be studied. We would combine the theory of polymer solution, single molecule force spectroscopy and molecular biology to analyze the structure-immunomudulatory activity with the help of laser light scattering and atomic force microspectroscopy. The relationship of chain conformation and immune activity would be elucidated..This work is useful to understand the mechanism of phellinus-igniarius polysaccharide biosynthesis. It will lay a theoretical foundation for directional preparation of high bioactive polysaccharide.

天然名贵食药真菌桑黄具有较强的免疫调节活性，其面临的野生资源匮乏瓶颈问题可通过液体发酵获得其次生代谢产物加以解决。近年来，通过添加真菌激发子等外源刺激物诱导食药真菌多糖类有效成分的合成已经成为真菌发酵调控研究的热点。前期的研究表明，真菌激发子可有效促进桑黄液体发酵多糖的含量和生物活性，然而关于其对桑黄多糖液体发酵合成调控的机理及其多糖空间构象的影响尚未见报道。基于此，本项目通过转录组技术比较在真菌激发子调控下的真菌RNA表达水平的变化，同时，测定合成途径中关键酶α-葡糖磷酸变位酶和葡糖磷酸异构酶的酶活变化，分别从分子水平上研究真菌激发子对桑黄多糖合成关键酶基因表达和蛋白表达水平的调控作用。同时，通过动静态激光光散射、原子力显微镜等技术对真菌激发子调控桑黄发酵并经纯化的活性多糖纯品FPIEPS-I进行精细结构及其空间构象的表征，以此阐明真菌激发子对高活性桑黄多糖生物合成调控的机理。

天然名贵食药真菌桑黄具有较强的免疫调节活性，其面临的野生资源匮乏瓶颈问题可通过液体发酵获得其次生代谢产物加以解决。桑黄多糖是桑黄发酵产物中的主要有效成分之一，桑黄多糖等食药用菌多糖是一种扶正固本的天然药物，被称为“生物应答调节剂”。获得多糖的分子链空间构象参数，可为进一步阐明分子结构与生物活性间的构效关系提供理论基础。项目利用桑黄菌株液体发酵技术获得桑黄胞外多糖，再通过生物柱层析技术，分别经DEAE-Sepharose Fast Flow离子交换色谱柱和Sephacryl S-100凝胶色谱层析分离纯化，得到均一多糖组分SHEPS2。通过光散射技术测定其分子量为9.43×104 Da。通过GC-MS测定其为甘露聚糖。再经IR、甲基化分析、核磁分析，发现SHEPS2是由以→3)-α-Manp-(1→为主链，→2)-α-Manp-(1→和→6)-α-Manp-(1→为支链的甘露聚糖。多糖的构象采用尺寸排阻色谱与光散射仪、示差检测器和粘度计（SEC-MALLS-IR-VIS）联用测定。结果显示多糖纯品SHEPS2的分子量（Mw）、流体力学半径（Rh）和特性黏度（[η]）分别为9.43×104 Da、8.09nm 和24.53 mL/g。其Mark-Houwink方程为[η]= 2.46×10-2 Mw0.58±0.006， Rh vs Mw关系的指数α、v分别为0.54和0.52，均表明多糖SHEPS2在0.1 M NaNO3溶液中为柔顺链构象。SHEPS2的蠕虫链模型构象参数为单位摩尔围长（ML）=453nm-1，持续长度（q）=1.04 nm，直径（d）=0.81 nm，表明多糖SHEPS2在0.1 M NaNO3溶液中为柔顺链构象。该多糖在溶液中的链构象经原子力显微镜（AFM）观测得到了进一步证明。本研究为阐明桑黄多糖SHEPS2的构效关系提供了有效的依据，为高活性多糖的定向制备奠定理论基础。