我国主要疯草内生真菌种群分布特征及遗传多样性研究

Locoweed is the major poisonous plant in the western grassland of China. Previous researches have shown that there are various fungal endophytes in locoweed, but their population distribution and inheritance are unknown. This project is based on our latest research results, fungal endophytes are isolated from the stems, leaves, flowers and seeds samples of five locoweeds Oxytropis kansuensis, Oxytropis glabra, Oxytropis ochrocephala, Astragalus variabilis and Astragalus strictus, distributed in different ecological regions of China. Fungal endophytes are isolated and purified from these locoweeds through the methods of surface disinfection and pointed hypha purification, species identification and phylogeny are analysed with morphological identification and rDNA-ITS sequence analysis, genetic diversity of fungal endophytes are carried out through RAPD and ISSR molecular marker technology, and DNA fingerprinting is constructed on fungal endophytes of major locoweed in China. Research Objectives is to investigate the genetic relationship on fungal endophytes of major locoweed from different species and regions and clarify species and distribution and genetic diversity on fungal endophytes of major locoweed in China. Research results are to provide a theoretical basis for the classification and rapid identification and genetic diversity of germplasm resources on fungal endophytes in locoweed, and to lay a foundation for researches on toxin synthesis and resistance mechanism and bioactive substances of fungal endophytes in locoweed.

疯草是我国西部草原的主要毒草。前期研究表明，我国疯草内含有多种内生真菌，而有关疯草内生真菌的种群分布特点及其遗传规律尚不清楚。本项目在已取得最新研究成果的基础上，以我国不同生态区域主要疯草（甘肃棘豆、小花棘豆、黄花棘豆、变异黄芪和茎直黄芪）为研究对象，采用表面消毒法和尖端菌丝挑取法分离纯化内生真菌，运用形态学和rDNA-ITS序列分析技术对其进行种属鉴定和系统进化分析，采用RAPD和ISSR分子标记技术对其遗传多样性进行研究，并构建我国主要疯草内生真菌DNA指纹图谱。旨在从分子水平探讨不同种类、不同地域疯草内生真菌的亲缘关系，弄清我国主要疯草内生真菌的种类及种群分布特征，揭示疯草内生真菌的遗传多样性及规律，为我国疯草内生真菌的分类与快速鉴定及其种质资源的遗传多样性研究提供理论依据，为疯草毒素合成与内生真菌互作关系、疯草内生菌与疯草抗性机制及疯草内生真菌生物活性物质研究奠定基础。

上世纪末，人们从疯草中分离到内生真菌，并初步认为疯草产毒机制与其内生真菌感染有关，随后证明了疯草内生真菌Undifilum oxytropis在疯草苦马豆素合成过程中起着主导作用。然而，有关我国疯草内生真菌的种类及优势菌株等尚不清楚。鉴于此，本项目选择了7个生态群落的4种疯草进行疯草内生真菌的分离培养与鉴定。采用表面消毒法和尖端菌丝挑取法分离纯化获得2194株内生真菌，分离率为50.9%。其中茎的分离率最高，叶其次，种子的分离率最低；运用形态学和rDNA-ITS序列分析技术对其进行种属鉴定和系统进化分析，结果显示，不同疯草其所含内生真菌种属不尽相同，但主要为Undifilum属、链格孢属（Alternaria sp.）、埃里砖格孢属（Embellisia sp.）、曲霉属（Aspergillus sp.）和镰刀孢属（Fusarium sp.）。其中Undifilum oxytropis普遍分布于疯草各组织中，是疯草内生真菌的优势菌株；采用RAPD和ISSR分子标记技术对疯草内生真菌的优势菌属Undifilum进行遗传多样性分析。结果表明，Undifilum属内生真菌遗传多样性较低，物种水平的遗传多样性显著高于种群水平。Undifilum属内生真菌的遗传变异发生于种群内部，种内存在一定的基因交流。通过本项目研究，基本弄清了我国主要疯草内生真菌的种类及种群分布特点，明确了Undifilum oxytropis是我国疯草内生真菌的优势菌株，支持了Undifilum oxytropis在疯草苦马豆素合成过程中起主导作用这一结论，为后续深入探讨疯草内生真菌Undifilum oxytropis的苦马豆素生物合成通路及动物疯草中毒病防控奠定理论基础。