基于多维组学方法的冬虫夏草子实体发育机制研究

Chinese cordyceps is a traditional Chinese medicine. The high price has made its excessive mining more serious, causing serious damage to the flagship species of Ophiocordyceps sinensis and its host ghost moth, as well as to the natural ecological environment on the Qinghai-Tibet Plateau. Artificial cultivation of Chinese cordyceps is the key way to resolve the above problems. However, the mechanism of formation of fruiting body, the key character of of O. sinensis, was not clear yet, which seriously restricted the rapid, efficient and eco-friendly development of the artificial culture industry of Chinese cordyceps. In this project, wild-type strain with complete life history and degenerated strains that cannot form fruiting bodies will be used to study the developmental process of O. sinensis from cadaver mummy (sclerotium) to fruiting bodies, paying particular attention to the mechanism of mycelium remodeling as the primordium of the fruiting body in the early stages of the formation of the fruiting body. Relying on the combination of high-throughput sequencing and gas chromatography-mass spectrometry, to carry out multidimensional omics analysis of comparative genomics, transcriptome and metabolomics, construct the gene regulation network of O. sinensis fruiting body development, obtain the differential gene expression profile of O. sinensis, reveal the key pathways and regulatory elements during the development of O. sinensis fruiting body, and clarify the molecular mechanism of the transformation of O. sinensis from anamorph to teleomorph in host. It is of great scientific and theoretical value to the studies of sexual development and phylogenetic evolution of O. sinensis and even entomopathogenic fungi. At the same time, the results of this study will provide theoretical and technical support for the artificial cultivation of fruiting body of O. sinensis, greatly promote the industrialization of Chinese cordyceps, and further contribute to the resources and ecological protection of Qinghai-Tibet Plateau.

居高不下的冬虫夏草价格使其过度采挖愈发严重，对青藏高原的旗舰物种冬虫夏草菌及其寄主蝙蝠蛾，以及自然生态环境造成了严重破坏。人工培养冬虫夏草是解决上述问题的根本途径，然而子实体形成这一关键性状的发生机制迄今尚不明晰，严重制约着其产业化发展。本项目拟利用具有完整生活史的野生型菌株以及子实体缺陷型菌株，聚焦于冬虫夏草菌侵染蝙蝠蛾幼虫后由僵虫至子实体形成这段时间的发育过程，特别关注子实体形成前期，菌丝体重构为子实体原基的过程机制，依托高通量测序技术与气质联用技术，进行比较基因组学、转录组学和代谢组学的多维组学分析，获得蝙蝠蛾虫体中冬虫夏草菌的差异基因表达谱，构建冬虫夏草菌子实体发育的基因调控网络，揭示其关键通路和调控元件，阐明冬虫夏草菌在寄主体内启动有性生殖的分子机制，对冬虫夏草菌乃至虫生真菌的有性发育及系统演化研究具有重要的科学意义和理论价值，并将为冬虫夏草子实体的人工培育提供理论与技术支持。

冬虫夏草是我国名贵中药材，虽已实现了人工培植，但子实体形成这一关键性状的发生机制尚不明晰，严重制约着其产业化发展。本项目聚焦于冬虫夏草菌侵染蝠蛾幼虫后由僵虫至子实体形成的发育过程，特别关注子实体原基的发育机制，进行比较基因组、转录组和代谢组的多维组学分析。通过单分子测序技术获取了冬虫夏草菌IOZ07菌株的近染色体水平的高质量参考基因组，大幅提升了基因组的连续性，可为昆虫病原真菌的遗传进化研究、种质资源保护与利用提供关键的遗传信息。子实体缺陷菌株1621在菌核发育阶段停滞，菌丝体的细胞融合过程受阻。与参考基因组IOZ07相比，1621菌株40个基因的编码产生了变异，其中细胞融合通路中的3个相关基因存在错义突变。在氧化胁迫下，退化菌株清除活性氧的能力更弱，由此建立了冬虫夏草菌人工培养菌种质量筛选的方法。利用转录组学和代谢组学技术，全面解析了子实体发育的不同时期冬虫夏草菌的基因诱导表达谱，以及代谢物的特征类群。研究显示，细胞膜的组分变化、氧化还原过程和离子的运输是贯穿子实体原基生长的三大主要因素，过氧化物酶体代谢通路及其相关联的脂代谢和氨基酸代谢是子实体原基发生期的主要营养代谢过程。通过对原基发生期的三个关键组织结构转录组分析发现，子实体原基为整个菌体的生长中心，菌核为能量代谢中心，菌毛的主要作用则是吸收环境中的营养物质，感知外界环境信号。调控有性生殖的重要信号转导蛋白COP9以及光感蛋白基因均在子实体的生长期和成熟期高表达，提示其对子实体有性生殖的调控始于子实体生长期，适当的光照处理是子实体形成的必要条件。基于转录组及代谢组信息，阐释了冬虫夏草重要活性物质虫草酸的生物合成途径。综上，通过多维组学分析，解析了冬虫夏草菌基因组及子实体发育的进程及其分子调控机制，阐释了子实体诱导发生期的营养代谢机制，对冬虫夏草菌乃至虫生真菌的有性发育及系统演化研究具有重要的科学意义，为冬虫夏草子实体的人工培育提供了理论支持与技术指导。