eno基因对蚕虫草和冬虫夏草子实体发育的影响及其分子机制

The fungus strain of Cordyceps militaris is often prone to degradation and resulting in sterile or deformed fruiting bodies on solid media, silkworm pupa or larva. Moreover, the fungus strain of Cordyceps (Ophiocordyceps) sinensis is also notoriously difficult to culture in vitro to form fruiting bodies. The reasons mostly attribute to the unclear molecular mechanism of fruiting body development. We systematically compared the differentially expressed proteins at different developmental stages of Cordyceps by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ ionization tandem time of flight (MALDI-TOF/TOF) mass spectrometry. Our results firstly showed the enolase expression is not only unanimously higher in fruiting body than in mycelium or stroma of C. sinenesis, C. militaris and C. memorabilis, but also is higher in late-stage fruiting body than that in early-stage. Recent studies have revealed that enolase not only participates in glycolytic cycle, but also plays important roles in pathogen infection. Besides，It is a multifunctional protein with unsuspected or unappreciated “second jobs”. Based on those evidences, we put forward a hypothesis that the enolase is not only a ubiquitous glycolytic enzyme involving in the glycolysis and glu-coneogenesis pathways that catalyzes the reversible dehydration conversion of 2-phosphoglyceric acid (2-PGA) into phospho-enolpyruvate (PEP) for supplying material and energy, but also prompts the fruiting body and spore development of Cordyces. In order to verify our hypothesis, RNA interference (RNA i) and gene over-expression technologies will be developed for investigating the effects of down-regulation and up-regulation of eno gene on fruiting body development of the silkworm C. militaris and C. sinensis. To clarify the effects of eno gene on fruiting body development and its mechanism of action determined, four levels of the changes in phenotype, mRNA level, protein level, and chemical level will be detected respectively by microscope, quantitative real-time PCR (RT-qPCR), Western blot and liquid chromatography tandem mass spectrometry (LC-MS). This project is benefit to reveal the molecular mechanism of fruiting body development and helpful to provide significant insight into the theoretical support for artificial cultivation of silkworm C. militaris and C. sinensis.

蛹虫草菌在固体培养和仿生培养过程中容易出现退化、畸形、不长子实体，冬虫夏草菌很难人工培养出子实体等问题，这些问题与人们对子实体发育机理不清密切相关。本项目组在研究冬虫夏草、蚕虫草、勿忘虫草不同发育阶段蛋白表达差异时，首次发现只有烯醇化酶 (Enolase) 在三种虫草子实体中的表达均显著地高于菌丝体或菌核（幼虫尸体），且晚期高于早期。近年报道Enolase不仅催化糖酵解反应，而且参与多种病理过程，表现出“兼职功能蛋白”的特点。因此，我们的假说是Enolase不仅具有催化活性产生磷酸烯醇式丙酮酸为子实体的构建提供基础，而且有助于虫草菌丝体由营养生长向生殖生长的转变，促进有性孢子的产生。为证实这一假说，我们拟用RNA干扰和基因过表达技术，从表型、基因、蛋白及其产物四个层次阐明eno基因对蚕虫草和冬虫夏草子实体发育的影响及其作用机制。本研究将为揭示虫草子实体发育的分子机理和人工培养提供理论支持。

从野生型冬虫夏草中多批次分离纯化得到其无性型—中国被毛孢（Hirsutella sinensis）并在固体培养基上成功培养出了冬虫夏草子实体，进一步证明了中国被毛孢是冬虫夏草真正的无性型，冬虫夏草子实体的形成完全可以不借助其他的共生菌或互生菌。从蛹虫草子实体中多批次分离纯化得到其无性型—蛹草拟青霉（Paecilomyces militaris）并成功在桑蚕幼虫、蛹和米饭培养基上进行仿生培养出蚕虫草（子实体生长在蚕幼虫上）蛹虫草（子实体生长在蚕蛹上）和虫草花（子实体生长在米饭培养基上），并完成了蛹虫草不同发育阶段的子实体的ATP含量测定。.完成了冬虫夏草和蛹虫草eno基因敲除载体骨架的筛选，基因敲除载体（OS-Enolase-Cas9和 CM-Enolase-Cas9）的构建；完成了冬虫夏草和蛹虫草Enolase基因过表达载体骨架的筛选，基因过表达载体（PCT-Enolase-OS和PCT-Enolase-CM）的构建。完成了冬虫夏草和蛹虫草原生质体制备和质粒转化条件优化。完成了冬虫夏草和蛹虫草Enolase抗体和内参抗体筛选（市面上没有这两个物种的Enolase抗体和内参抗体）。完成了蛹虫草Enolase基因过表达载体的转染，并获得了单克隆菌株，菌丝体生长状况优于野生型对照组。完成了蛹虫草Enolase基因敲除载体的转染，并通过Western Blot检测显示基因敲除后的菌株Enolase的蛋白含量显著低于野生型对照组，菌丝体生长不良。该研究证明eno基因对虫草发育至关重要，同时对冬虫夏草和蛹虫草的选育具有重要的参考意义。.额外完成了冬虫夏草不同发育阶段的转录组高通量测序分析，获得了冬虫夏草不同发育阶段的转录组数据，一共获得了85463个转录本，获得了不同发育阶段样本间差异表达基因和其参与的403个信号通路，以及差异表达基因的功能富集和功能关联情况。额外完成了蛹虫草不同发育阶段的转录组高通量测序分析，获得了蛹虫草不同发育阶段的转录组数据，一共检测到9652个已知转录本和10898个新转录本，获得了不同发育阶段样本间差异表达基因和其参与的370个信号通路，以及差异表达基因的功能富集和功能关联情况，同时还获得了630个高可信度的lncRNAs和其调控的靶基因，所有的Raw Data均已上传共享数据库，对于冬虫夏草和蛹虫草基因功能和作用机制的研究具有重要参考价值。