应用转录组和蛋白组学技术挖掘名贵中药冬虫夏草侵染后形成子座的关键基因与蛋白质研究

Artificial cultivation of Cordyceps sinensis as a research hotspot and difficulty, a breakthrough in recent years, but the formation rate of fruiting bodies after infection is low has always plagued its difficult problem of efficient cultivation. The study on the formation mechanism of infective fruiting bodies after infection has also become the key to efficient cultivation of Cordyceps sinensis, and there are few studies. The research team found that 35 new protein spots appeared after the formation of fruiting bodies, which may be related to fruit body formation. A significant number of fruit body development-related gene expressions were upregulated after transcriptome formation. It suggests that key genes and proteins formed from fruit bodies may be excavated from the proteome and transcriptome levels. The transcriptome combined protein group has not been reported to investigate the mechanism of the invasive fruit body formation of Cordyceps sinensis. Based on the previous study, this study plans to deeply study the transcriptome changes before and after the infestation of infective fruiting bodies, combining the results of previous studies to find out the changes in related genes of fruiting bodies, screening key genes for the formation of fruiting bodies, and using protein proteomics to reveal these stages of Cordyceps sinensis. Fruit body-related protein changes, screening for protein markers, integration analysis of both protein groups and transcriptomes, identification of key genes and proteins, verification by knockout, revealing the key gene and protein signatures of fruiting bodies infested by Cordyceps sinensis The object will lay a good foundation for the standard and efficient cultivation of Cordyceps sinensis.

冬虫夏草人工培育作为研究热点和难点，近年来突破较大，但侵染后子实体形成率低一直是困扰其高效培育的难题。侵染后子实体形成机制研究也成为冬虫夏草高效培育的关键，目前研究较少。课题组发现：子实体形成后出现了35个新的蛋白质斑点，可能与子实体形成相关；转录组发现子实体形成后有较多子实体发育相关基因表达上调。提示从蛋白组和转录组水平可能挖掘到子实体形成的关键基因和蛋白质。转录组联合蛋白组研究冬虫夏草侵染子实体形成机制未见报道。本研究拟在前期基础上，深入研究侵染后子实体形成前后的转录组变化，结合前期结果寻找子实体相关基因变化情况、筛选子实体形成的关键基因；同时应用蛋白组技术揭示冬虫夏草这些阶段子实体相关的蛋白质变化，筛选蛋白标志物，将蛋白组和转录组二者整合分析，明确关键基因和蛋白质，通过基因敲除进行验证，揭示冬虫夏草侵染后子实体形成的关键基因和蛋白标志物，将为冬虫夏草规范高效人工培育奠定良好的基础。

冬虫夏草Ophiocordyceps sinensis由于其生理生化的特殊性和复杂性，目前其子实体形成机制研究得并不明确，影响了其野外恢复与人工培育工作。. 本研究顺利完成了冬虫夏草转录组测序、蛋白组分析。其中转录组测序、生物信息学分析与筛选子实体58个差异表达基因下调，103个差异表达基因上调基因，表明冬虫夏草蛋白质合成的基本代谢在冬虫夏草菌感染小金蝠蛾幼虫后发生了改变，以及47个基因特异表达；还发现肌动蛋白和微管蛋白相关基因表达的改变可能在子实体发育中起作用。利用蛋白组技术对冬虫夏草的发育过程中的三个阶段（菌虫复合体、子座、整体）的蛋白质组进行分析，筛选出5个稳定存在的蛋白质和3个具有显著差异的蛋白质，这8种蛋白质均在子座形成后出现。最终提示肌动蛋白可能在冬虫夏草的生长发育中其重要作用。综合二者分析发现肌动蛋白可能在冬虫夏草子座形成中起重要的作用，后续验证也证实了这一结果。. 对比野生冬虫夏草与人工冬虫夏草转录组与蛋白组分析发现：野生与人工冬虫夏草差异表达基因显著性富集 到直接或间接与脂肪酸代谢相关途径， ACAD 、ECH 、KAT 、atoB 等关键酶在野生冬虫夏草中表达量更高。转录因子分析表明， 野生冬虫夏草在响应非生物胁迫时，可增强各转录因子的表达，从而增强下游响应胁迫基因表达以适应逆境生长条件。 使用iTRAQ 技术对冬虫夏草进行蛋白组学分析，再与转录组结果联合分析。关联后的共表达模式内的多数差异蛋白参与脂肪酸代谢相关的生物途径、糖酵解糖异生能量代谢以及调节机体对低氧浓度和紫外线辐射应答通路，这在一定程度上亦能验证转录组结果，从蛋白水平上揭示了造成野生冬虫夏草与人工冬虫夏草差异分子机制。通过 qRT-PCR、MDA 和PRO生理指标测定、GC-MS 检测野生人工冬虫夏草中脂肪酸成分含量等方法证实了脂质代谢与抗逆性直接相关。. 本研究初步揭示了冬虫夏草形成的关键基因和蛋白，将为后续冬虫夏草精准野外恢复与人工培育提供技术支撑。