西藏冬虫夏草遗传多样性及与虫草品质相关性研究

Ophiocordyceps sinensis is one of rare and precious Chinese herbal medicines. In the O. sinensis producing areas of the Tibet Autonomous Region, more than 80% of farmers incomes were from the O. sinensis. Quality of O. sinensis which was produced in Tibet, is dissimilarity due to the differences of "provenance" and "land races". But until now, the quality evaluation of O. sinensis is still based on the external morphological characteristics. There is no a specific, scientific and convenient evaluation method using for quality evaluation. The objective of this project is that carry out study of the correlational between quality and genetic diversity of O. sinensis, as well as between its host insects, using the means of morphology, phytochemistry, molecular biology. The study will be on the effecting of environment, genetic variation of O. sinensis and host insect on activity compositions of O.sinensis, also clarify the quality characteristics and influencing factors of O.sinensis which produced in Tibetan. It is building a technology platform and providing theoretical support to make a scientific system for evaluating quality of O.sinensis, which is very important to deeper study and economic development of Tibetan O.sinensis.

冬虫夏草是我国珍稀名贵中药材，西藏自治区虫草产区农牧民收入的80%以上来自于冬虫夏草。西藏冬虫夏草因其"地源"和"种源"的不同，品质差异较大，但目前其品质仍以外部形态特征来评价，缺少客观、科学和便捷的评价方法。本项目综合运用形态学、植物化学、分子生物学等手段，开展西藏冬虫夏草遗传多样性与冬虫夏草品质相关性研究，分析冬虫夏草菌及寄主蝙蝠蛾的遗传变异、产地环境等对西藏冬虫夏草有效活性成分的影响，阐明西藏不同"种源" 和"地源"冬虫夏草的品质特点及影响因素，筛选与冬虫夏草品质相关的特征性指标，构建科学的冬虫夏草质量评价体系，为科学、准确地鉴定冬虫夏草的等级、真伪及其品质提供理论支持和技术平台，对西藏地区冬虫夏草资源的利用与深度开发具有重要意义。

1、西藏地区采集110份冬虫夏草样品，分离获得60个冬虫夏草菌。不同产地冬虫夏草菌菌落培养性状上稍有差异。基于10个基因序列的冬虫夏草遗传多样性和种内变异分析表明，不同产地冬虫夏草具有丰富的遗传多样性，67份样品可以分为5个不同clade, 且每个clade,有明显的地域分布特征。.2、发现并报道Pochonia cordycepisociata H. Huang, M. Wang & L Cai, sp.nov.新种1个。.3、通过 HPLC 法检测了我国 24 个冬虫夏草居群的子座和虫体中主要核苷类成分及含量。结果表明冬虫夏草不同部位的核苷类成分的组成和含量存在明显差异。冬虫夏草子座中含有尿苷、鸟苷和腺苷 3 种核苷类成分，其中腺苷含量最高，尿苷次之，鸟苷最低。虫体中含有胞苷、尿苷、肌苷、鸟苷和腺苷 5种核苷类成分，其含量高低顺序为鸟苷>尿苷>腺苷>肌苷>胞苷。LSD 分析结果表明冬虫夏草子座的 3 种核苷类成分含量及核苷总量、虫体的 5 种核苷类成分含量及核苷总量在 24 个冬虫夏草居群间均存在显著差异。冬虫夏草子座和虫体中尿苷、鸟苷和腺苷的含量均较高，可以考虑增加尿苷、鸟苷这 2 个指标与腺苷一起作为冬虫夏草的质量控制因子。.4、对我国青藏高原冬虫夏草5个主要产区的50个冬虫夏草居群，的33个冬虫夏草菌的rDNA ITS 基因和β-tubulin基因以及其寄主昆虫的线粒体COI、COII和Cytb基因片段序列进行分析，基于5个基因序列分析的冬虫夏草菌和寄主昆虫的变异参数和碱基组成、冬虫夏草菌和寄主昆虫的遗传距离和地理距离相关性分析、冬虫夏草菌与其寄主昆虫单倍型多样性以及分支间遗传分化分析表明rDNA ITS基因与COI基因被选为分析冬虫夏草菌与其寄主昆虫系统发育关系的最适分子标记。系统发育分析、Mantel检验、AMOVA分析均显示冬虫夏草菌及其寄主昆虫有着相似系统发育关系、地理分布格局和遗传分化，初步推测冬虫夏草菌与其寄主昆虫发生了协同进化。