苦荞内生真菌对宿主黄酮类成分合成积累的调控效应及机制研究

Tartary buckwheat is a pronounced medicinal and edible coarse cereals, and flavonoid is the most important functional component of tartary buckwheat along with its products. China is the largest tartary buckwheat-producing country in the world. It is very important to effciently control and enhance the flavonoid content for promoting the nutritional and economical value of tartary buckwheat. Plant endophytic fungi is an important and novel microbial resourse, and shows great potential applications in promoting the growth, and regulating secondary metabolisms biosynthesis of its host plant. Based on previous investigations, the tartary buckwheat is aboundant in fungal endophytes, and the crude polysaccharides of some fungal isolates could effciently stimulate the flavonoids accumulation (increased by more than 35%) of their host plant, especial for the endophytes of Bionectria sp. Fat-6 and Fusarium sp. Fat-9. This study will be mainly focused on isolation and identification of the bioactive polysaccharides and oligosaccharides from the objective fungal endophytes by bioassay-guided fractionation. Furthermore, the stimulation effects of polysaccharide or oligosaccharide on flavonoids biosynthesis and accumulation of tartary buckwheat, as well as their prelimary regulation mechanism would be clarified in detail. Through this research, the application of specific fungal elicitors could be a new efficient strategy for improving the flavonoids content and quality of tartary buckwheat, and more beneficial for the development of buckwheat industry in our country. Meanwhile, it could provide more scientific basis for illustrating the relationship between endophytic fungi and their host plant.

苦荞是一种著名的食药两用特色杂粮作物，黄酮类成分是苦荞及加工制品发挥药用保健功效的重要物质基础。我国是世界上苦荞种植面积和产量最大的国家。在苦荞种植生产过程中，如何有效控制提高黄酮类功能成分含量对于提升苦荞的营养保健及经济价值至关重要。植物内生真菌生境独特，在促进宿主植物生长及次生代谢物合成积累等方面具有巨大潜能。前期研究表明，苦荞内生真菌资源丰富，部分内生真菌粗多糖能够有效提高苦荞黄酮含量35%以上，其中以生赤壳霉Fat-6和镰孢菌Fat-9的促进效应最为明显。在此基础上，本项目拟采用活性追踪法分离纯化目标内生真菌中的活性多糖及寡糖，并解析其化学结构特征；进一步阐明内生真菌多糖及寡糖对苦荞黄酮类成分合成积累的调控规律及初步作用机制。本项目的突破将为提高苦荞黄酮含量及品质提供一条新途径，有助于促进苦荞产业健康发展，同时也为丰富和发展现有的内生真菌调控宿主植物次生代谢理论提供新的科学依据。

苦荞是一种著名的药食两用特色杂粮，具有很好的营养保健价值。植物内生真菌是一类重要的微生物资源，能产生多种活性成分，在促进宿主生长及次生代谢物合成等方面具有巨大潜能。本项目围绕“利用内生真菌及其活性物质提高苦荞麦中黄酮类成分含量累”这一主题，通过三年的研究，从苦荞内生真菌中成功分离制得3种活性多糖，明确了其初步化学结构特征，进一步阐明了活性多糖促进苦荞芽黄酮类成分合成积累规律及诱导机制等，主要结论如下：（1）通过液体悬浮培养和水提醇沉法等方法获得内生真菌Fataf6胞外多糖（EPS）、菌丝水提多糖（WPS）和碱提多糖（SPS）3种粗多糖，其中EPS和WPS对苦荞芽黄酮合成具有较好的促进作用；进一步采用DEAE-52纤维素柱层析方法分别对EPS和WPS进行分离纯化，共获得3个主要多糖EPS-I、WPS-I和WPS-II；通过HPLC法对3种多糖的组成进行分析，发现其主要由葡萄糖、半乳糖和甘露糖组成；（2）EPS-I、WPS-I和WPS-II对苦荞芽生长和黄酮类物质合成具有较好的促进作用，其诱导效应与多糖的类型和处理浓度紧密相关，其中以EPS-I的促进作用最为显著。当EPS-I的处理浓度为150 μg/mL时，苦荞芽中主要黄酮类物质荭草素、异荭草素、牡荆素、异牡荆素、芦丁和槲皮素分别为0.18 mg/g、0.32 mg/g、1.03 mg/g、1.51 mg/g、49.09 mg/g和1.23 mg/g，分别较对照提高了20.00%、68.42%、39.19%、17.97%、21.57%和95.23%；（3）苦荞芽经活性多糖EPS-I（150 μg/mL）、WPS-I（50 μg/mL）和WPS-II（100 μg/mL）处理后，其诱导周期内苦荞芽培养液中pH值与电导率，苦荞芽中PAL、PPO和SOD活力，黄酮类成分含量，以及苦荞黄酮合成途径中关键酶基因（FtPAL、FtC4H、Ft4CL、FtCHS、FtF3H和FtF3’H等）表达量等指标较空白对照组均有显著增加。推测内生真菌Fataf6活性多糖（如EPS-I）能有效激发苦荞芽的防御反应，产生的相关诱导信号（如H+等）可通过信使分子进行逐级传递和放大，从而引起苦荞（芽）黄酮合成途径中的特定功能基因的高效表达，激活关键酶活力，进而促进苦荞芽中黄酮类次生代谢产物的合成积累。本研究结果为提高苦荞黄酮含量及品质提供一条新途径。