珍稀野生食药用菌蒙古口蘑基因组谱系与功能相关性研究

Tricholoma mongolicum S. Imai mainly distributes the Inner Mongolia grassland of China. It is an endemic fungus of the Mongolia Plateau. Its nourishment and medicinal value have owned a great reputation among the local people of all ethnic groups. As a result of environmental deterioration and indiscriminate digging, its resources are in an endangered state. It is imminent to carry out researches on genetic diversity conservation and sustainable utilization on T. mongolicum. The project will be conducted on research mode “Resource conservation - functional gene - active component - molecular mechanism”. Functional genes will be obtained by genome screening that has contributed to goal-oriented natural active components extraction. The whole genome of T. mongolicum will be sequenced by de novo and full length transcriptome sequencing based on third generation sequencing technology. The complete genome of T. mongolicum will be analyzed with Bioinformatics and the functional genes and metabolic pathways related the active ingredients of T. mongolicum will be screened. The authenticity of the functional genes related to the metabolism of active ingredients will be verified by designing specific primers and with the method of Real-time Quantitative PCR, Western Blot and Metabolomics. Appropriated animal models will be used to observe the effect of characteristic components of T. mongolicum on strengthening immunity and anti-tumor. The molecular mechanism responsible for the biological activity of active components will be summarized. The fermentation process of the maximum effective component will be obtained by which optimal fermentation conditions are studied to stimulate the efficient expression of functional genes. The germplasm resources will be established based on T. mongolicum genome information and provide the basis for its conservation and utilization in the future. The project will provide exactly basic research results for the further development and utilization of T. mongolicum. It will play a model role in deep development to use endangered wild mushroom resource.

蒙古口蘑主要分布我国内蒙古草原，是蒙古高原特有真菌，其滋补和药用价值在当地各族民众中自古享有盛誉。由于环境恶化及滥采滥挖，资源处于濒危状态，开展遗传多样性保育和可持续利用研究迫在眉睫。本项目采用“资源保育—功能基因—活性成分—分子机理”整体性的研究模式，对蒙古口蘑进行全基因组的de novo测序与基于第三代测序技术的全长转录组测序；对蒙古口蘑全基因组进行生物信息学分析，筛选出蒙古口蘑中活性成分相关的调控基因和代谢通路；通过设计特异性引物、采用实时荧光定量PCR与Western Blot及代谢组学等方法对活性成分代谢相关的基因的真实性验证，并依据注释通路设计提取成分方案；获得该真菌的特征性成分和抗肿瘤与增强免疫活性成分，并阐明活性分子机理。通过研究最优发酵条件，激发高效的功能基因表达，获取最大化的有效成分发酵培养工艺，建立以蒙古口蘑全基因组信息学为基础的种质资源库，为保育和利用提供基础。

蒙古口蘑是蒙古高原特有珍稀物种，因其资源濒危，亟待开展保育和可持续利用系统研究。本项目采用“资源保育—功能基因—活性成分—分子机理”整体性模式对该菌进行了研究。全基因组的de novo与全长转录组测序，首次获得蒙古口蘑全基因组信息，基因组大小为45.98Mb，保护26个组装片段，contig N50长度为3.6Mb，预测到11599个基因；碳水化合物酶相关基因在子实体形成过程中起重要作用，该菌有基因丢失现象。全基因组分析发现384个碳水化合物酶相关基因；基于两条KEGG有42个基因注释到甾类和萜类合成通路；甾醇24-C-甲基转移酶与异戊二烯半胱氨酸羧甲基转移酶基因在两条通路中有高表达；子实体阶段的甾类与萜类合成基因并无特异表达。获得了高多态性的该菌的SSR分子标记8个；基于基因组发现1821个分子标记位点。化学成分研究表明该菌为高蛋白、高氨基酸含量的珍贵食药用菌。应用HPLC等方法从子实体得到以环戊烷骈多氢菲为母核，侧链的C24上有甲基的甾类化合物8个。本研究证实了蒙古口蘑显著的抗肿瘤、增强免疫及催乳作用，其中石油醚提取物和甾类成分效果显著，其抗肿瘤机理主要通过对色氨酸代谢、5-羟色胺能突触、氨基酸的生物合成与花生四烯酸代谢等多个代谢通路和对肠道微生物影响、增强免疫、促进细胞凋亡以及抑制肿瘤血管新生等多种途径。甾类成分能激活巨噬细胞的吞噬能力，4,6,8（14）,22（23）-四稀3-酮-麦角甾烷的效果最佳。石油醚提取物和水提取物通过上调乳腺组织中α-乳白蛋白、β-酪蛋白、催乳素受体、信号转导和转录激活因子5、雌激素受体和孕激素受体的转录起到显著的催乳作用。确定了蒙古口蘑液体发酵和固体发酵最佳条件，发酵菌丝体可以代替子实体应用。本研究成果为该菌的保护及可持续利用提供了研究基础，确定甾类成分是其特征性和活性成分。该菌对某些甾类和萜类代谢酶的高表达特性与富含多种甾类成分及甾类显著的药理活性，三者具有高度的一致性，本成果在蒙古口蘑及其它菌物药资源的可持续利用和新产品开发利用方面具有重要价值和意义。