含毒蕈碱和异噁唑衍生物类有毒蘑菇的毒素成分及系统发育研究

In recent years, with the extensive investigation of mushroom poisoning in China, more and more cases of nerve mushroom poisoning have been occurred, which were caused mainly by the species of Inocybe and Amanita genus section Amanita containing muscarine and isoxazole derivatives. The research on resources, morphological classification and molecular phylogeny of this kinds of mushrooms have made good achievements. However, the detection and study of toxins in this kinds of mushrooms is completely blank in our country. Based on the previous research progress, this project intends to carry out four aspects of research: (1) to collect the samples of Inocybe and Amanita genus section Amanita and to establish phylogenetic tree based on multi-locus DNA sequence; (2) to establish the qualitative and quantitative methods of muscarine and isooxazole derivatives by means of HPLC methods, and to analyze the toxin components and their contents of mushrooms in China; (3) to identify the toxins and relative metabolites and to establish the fingerprints of toxins and relative metabolites of mushrooms based on HPLC-MS and hierarchical cluster analysis was carried out. (4) to study the relationship between the phylogenetic tree based on toxin component and the phylogenetic tree based on multi-locus DNA sequence. The implementation of this project will greatly enrich the species and components of poisonous mushroom in China. It is of great scientific significance to understand the toxin diversity and chemical classification of the two genera, and has important application guiding value for the prevention of mushroom poisoning, diagnosis and treatment of mushroom poisoning in China.

近年来随着对我国蘑菇中毒事件的广泛调查和深入，导致神经中毒的事件越来越多，主要由含毒蕈碱和异噁唑衍生物的丝盖伞属和鹅膏菌属鹅膏组的一些种类所致。在我国，对于这类蘑菇的资源、分类和系统发育研究取得了一定成绩，但是对于这类蘑菇的毒素检测和研究则完全处于空白状态。在前期研究的基础上，本项目拟开展4个方面的研究：（1）通过中毒调查、野外采集收集丝盖伞属和鹅膏菌属鹅膏组标本，进行分子系统发育研究；（2）利用HPLC技术建立毒蕈碱和异噁唑衍生物定性定量方法，检测各物种的毒素成分及含量；（3）利用HPLC-MS鉴定各物种的毒素及相关代谢成分，并依据HPLC指纹图谱进行聚类分析，构建系统树；（4）探讨基于毒素和相关代谢成分的系统树和基于DNA序列的系统树之间的关系。该项目的实施将极大丰富我国毒蘑菇种类及毒素成分，对于了解毒素多样性、化学分类具有重要科学意义，对于我国蘑菇中毒预防和诊断治疗具有重要指导价值。

近年来，神经精神型蘑菇中毒事件在我国频繁发生，这类事件主要因误食含毒蕈碱的丝盖伞科（Inocybaceae）和含异噁唑衍生物的鹅膏菌属鹅膏组（Amanita section Amanita）的野生蘑菇所引起。在我国，以前对于这类蘑菇中的有毒蘑菇种类多样性和毒素成分缺乏深入研究。本项目从形态学、分子系统学和毒素成分等方面对丝盖伞科和鹅膏属鹅膏组物种展开了系统研究。研究结果表明：（1）在物种多样性和物种鉴定方面，共采集和整理了丝盖伞科标本395份，鹅膏属鹅膏组标本146份，丝盖伞科物种经形态和分子鉴定，确定有205个分类单元，分属于丝盖伞属，歧盖伞属，茸盖伞属和裂盖伞属，已鉴定62个物种，其中36个为中国新记录种，已发表6个新种和1个中国新记录种；鹅膏属鹅膏组物种确定为28个物种，已发表2个新种。（2）在毒素检测方面，通过超高效液相色谱串联质谱（UPLC–MS/MS）法对收集的材料进行毒素检测，结果表明165个检测的丝盖伞科物种中91个物种含毒蕈碱，其中新发现丝盖伞科有毒种23个，74个物种未检测出毒蕈碱。20个鹅膏属鹅膏组检测物种中10个物种含有鹅膏蕈氨酸和异鹅膏胺，10个物种不含这两种毒素。（3）在系统发育方面，基于5个DNA位点（ITS，nrLSU，RPB2，RPB1和TEF1-α）重建的丝盖伞科系统发育树有4个分支，分别为丝盖伞属（Inocybe），茸盖伞属（Mallocybe），岐盖伞属（Inosperma）和裂盖伞属（Psuedosperma）；其中毒蕈碱主要分布于丝盖伞属，裂盖伞属和岐盖伞属中的斑点组（Maculatum clade）。基于5个DNA位点（ITS，nrLSU，RPB2，TUB2和TEF1-α）重建的鹅膏属鹅膏组系统发育树分为4个主要分支，含有毒素的物种大多数分布在其中两支；鹅膏属鹅膏组基于次生代谢产物构建的系统发育树与基于ITS构建的系统发育树基本一致。该项目所获得的研究结果弄清了我国丝盖伞科和鹅膏菌属鹅膏组的毒蘑菇种类和毒性，探究了这类蘑菇的系统分类、化学分类、毒素起源等问题，可为我国毒蘑菇中毒预防、诊断治疗提供科学指导。