通过转录组分析研究冬虫夏草菌子实体发育启动的关键基因

A experimental system, created by auther, in which the fruit bodies of Ophiocordyceps sinensis can be obtained constantly, would be used in this study. It would be set a treatment that fruit body development has been triggered and produce fruit bodies, a control that stay in vegetative growth phase. Tissue materials would be samplinged from two groups to extract mRNA in certain time and the cDNA would be synthesized by inverse transcriptase. Meanwhile， cytolgical examination and morphological investigating would be implemented. The cDNA Illumina libraries which have genes triggering the fruit body development would be set up. Those genes that related to the the fruit body development of the fungus would be found by analyzing and comparing with database system. It would be investigated the continuous change in genes and mycelium growth, the relationship between gene express and morphologic change by pomparing different periods of vegetative growth, the fruit body development and growth. It would also expelore the molecular mechanism in the fruit body development, including: external environment, genes transcription, protein express，cell differentiation and morphogenesis in detail. It would reveal the relationship between environment, genes and morphological differentiation in Ophiocordyceps sinensis initially. It would provide a helpful basis for further research and exploit the usage of this fungus resourse.

申请人已经建立了人工培养条件下形成冬虫夏草菌子实体的实验体系，通过设立对照组和促子实体形成（出草）处理组，定期取样，对菌体进行形态学和细胞学显微观察记录，同时分别提取对照组与处理组菌体mRNA，反转录合成双链cDNA，构建Illumina文库，桥式PCR，Illumina PE测序，生物信息学分析等方法，筛选出与该菌子实体发育启动密切相关的关键基因。通过对营养生长期、子座形成期、子实体生长期等关键节点上，该真菌在菌丝形态学、细胞学和转录基因的相互比较，揭示冬虫夏草菌子实体发育启动相关的关键基因及其动态变化，以及与之相对应的细胞学和形态学特征方面的对应关系。探索该真菌子实体发育过程中：环境变化--基因转录--蛋白表达--形态建成的变化机理。初步揭示冬虫夏草菌子实体发育启动阶段的环境-基因--形态相互对应的关系。为今后更好地研究和利用该宝贵资源提供一定基础。

本项目主要针对冬虫夏草菌中国被毛孢子实体发育启动前后分别提取对照组与处理组菌体mRNA，反转录合成双链cDNA，构建文库，通过基因表达的差异来寻找关键基因。利用Trinity软件自体组装共获得203,619条Unigenes。其中，两种组织共有的Unigenes为141,254条，发育前样品特有的Unigenes为28,541条，发育后样品特有的Unigenes为33,824条。在子实体发育前后两个时期样品各自特异性表达61和73条基因。两个时期样品中共同基因中，有1,298条基因表达差异显著(&nbsp;|log2(FoldChange)| > 1 & P-value < 0.05）。其中，发育前相较于发育后有410条Unigenes表达显著上调，而发育后相较于发育前有888条基因表达显著上调。研究发现长度为957bp蛋白激酶Hippo（Hpo）基因和29k 的Rac-1基因在子座发育中表达呈极显著的上调，是冬虫夏草子座发育的关键基因。低温处理后出现差异表达的基因分析发现：ggt基因和sdh基因分别为两个通路下的关键基因，fmid和ghf3基因以及tpi和rpi基因为关键基因上游或下游的顺序基因。在此基础之上构建了基因顺序表达的假定模型。同时应用实时定量PCR技术对转录组数据的可靠性进行了验证。项目实施中从冬虫夏草上分离发现了一些与冬虫夏草有关系的菌种，被称为“冬虫夏草相关菌”。对得到的7个菌株并进行了初步研究，发现其均可以感染蝙蝠蛾幼虫，并与中国被毛孢存在营养竞争关系。通过对低温处理140d后取样进行扫描电镜观察可以看出，实验组处理中可见较为明显的菌丝扭结现象，与之相比较对照组中未见明显的菌丝扭结及融合现象。