内生真菌DO14促铁皮石斛多糖成分积累的作用机制

Dendrobium officinale is ranked “the first of the nine Chinese fairy herbs”. Water-soluble polysaccharides are its main ingredients, which exhibit anti-inflammatory, immuno-enhancing, anti-tumor and other activities. Our previous studies had found that endophytic fungi Pestalotiopsis sp. DO14 isolated from the stem of D. officinale could significantly improve the content of polysaccharides in the stem. Therefore, this study will focus on how the fungus DO14 affects the polysaccharides accumulation. Sterile clonal tissue-culture seedlings of D. officinale and the seedings co-cultured with DO14 will be studied as subjects. The impact of the target strain DO14 on the regularity of polysaccharides changes in D. officinale plantlets will be revealed by using HPLC-MS saccharide mapping combined with metabonomic technology. Also, the impact of DO14 on the growth and physiological characteristics of D. officinale plantlets that related to the changes of polysaccharides will be clarified by observing microstructure and determining photosynthetic physiological index of D. officinale plantlets. And the transcriptomic technology will be applied to find the key enzyme genes involved in polysaccharides biosynthesis. Then, the mechanism that endophytic fungi DO14 affect polysaccharides of the host plant will be explained. This study is intend to provide scientific evidence for revealing the mechanism that endophytic fungi affect the quality of the medicinal plants, and also has important significance to improve the quality of D. officinale.

铁皮石斛位列中华九大仙草之首，水溶性多糖为其主要功效成分，具有抗炎、增强免疫、抗肿瘤等药理活性。课题组在铁皮石斛品种选育及栽培研究中发现，从铁皮石斛茎中分离的内生真菌Pestalotiopsis sp. DO14能极显著提高茎中水溶性多糖的含量。本项目以内生真菌Pestalotiopsis sp. DO14为研究材料，以接DO14菌与无菌铁皮石斛无性系组培苗为研究对象，利用HPLC-MS糖谱法和代谢组学技术揭示DO14对铁皮石斛组培苗多糖成分变化的影响规律，结合铁皮石斛组培苗形态解剖、光合生理特性研究，明确在DO14作用下与多糖变化相关的生长生理特性，同时利用转录组学挖掘DO14作用下与铁皮石斛多糖变化相关的关键酶基因，从而阐释内生真菌DO14促铁皮石斛多糖成分积累的作用机制，为揭示内生真菌影响铁皮石斛品质及其机制提供理论依据，对铁皮石斛药材品质稳定和提升具有重要意义。

铁皮石斛位列中华九大仙草之首，水溶性多糖为其主要功效成分，具有抗炎、增强免疫、抗癌等药理活性。课题组前期研究发现铁皮石斛茎中分离的内生真菌Pestalotiopsis sp. DO14能极显著提高茎中水溶性多糖的含量。为揭示铁皮石斛内生真菌DO14促宿主多糖积累的变化规律及作用机制，本项目通过对不同培养时间点铁皮石斛接DO14菌和无菌无性系组培苗形态特征和解剖结构观察，光合生理特性、多糖含量及糖类组分测定，转录组测序分析，得到如下主要结果：（1）明确了DO14侵染宿主的过程及其对宿主生长的影响：DO14菌丝主要分布于铁皮石斛根表面于第3-6天侵入根被细胞内并定殖，第8天在部分根被细胞内形成菌丝团，充满菌丝的细胞中淀粉粒或叶绿体消失；与DO14共培养第8天后，茎中部粗及生物量均极显著提高，第15天后茎、叶开始变紫红色；（2）揭示了DO14侵染对宿主生理生化的动态影响：DO14侵染后宿主光合活性、光能转化效率与光保护能力明显改变，而叶绿素a、叶绿素b、叶绿素a/b及叶绿素总量减少或无明显变化；叶、茎中SPS酶活都显著增加，3-15天茎中蔗糖和果糖含量显著增加，随后下降；第3天后叶中单糖、二糖、已糖类成分大量积累；第6天后茎中多糖含量显著增加，甘露糖与多糖变化趋势一致；（3）挖掘出与铁皮石斛糖类代谢途径相关的关键差异基因，探讨了DO14促铁皮石斛多糖积累的分子作用机制：DO14侵染后，铁皮石斛叶和茎中PSⅠ、PSⅡ和电子传递系统相关的多数差异基因显著上调，糖代谢通路中蔗糖、果糖、葡萄糖、甘露糖积累相关的差异基因（RBCS、GAPC、FBA、SPS1/2、SPP2、IVR1、CIN3/7、GLU1、BGLU、GLC1、MAN1/6和FRK2等）表达显著上调，同时编码糖转运蛋白基因STP11/13、SWEET14/15及参与葡甘聚糖合成基因CSLA9显著上调，最终促使铁皮石斛茎中多糖（以葡甘聚糖为主）积累。研究结果验证了项目提出的科学假设，为揭示内生真菌影响铁皮石斛品质及其机制奠定基础，对铁皮石斛药材品质稳定和提升具有重要意义。