蛹虫草光受体CmWC-1调控子实体发育及类胡萝卜素合成机制的研究

Cordyceps militaris (L.) Link is one of edible and medicinal fungi with high nutritional value and tonic activity. Light is the key factor for the fruit body development and carotenoid production. Our previous study has confirmed that blue light acceptor CmWC-1 is a nessssary transcript factor for the fruit body development and carotenoid production by gene disruption in Cordyceps militaris. However, the mechanism is not clear. In the present study, a whole-genome gene expression analysis was performed with the wild-type and the Cmwc-1 deletion mutant grown under dark or light conditions to forecast the target gene of this transcript factor. The direct target gene of CmWC-1 and the binding DNA motif was then identified by chromatin immunoprecipitation assay (Chip). The transcript factors and genes related to fruit body development and carotenoid production were found among the target genes of CmWC-1. The relative expression of these genes were analysised and gene disruption was performed to study the functions. The realtion between these genes, tracnscript factors and CmWC-1 will be showed and the regulation mechanism of CmWC-1 on fruit body development and carotenoid production will be presented. This study will lay a foundation for the study on light reaction mechanisms and the molecular basis of the fruit body formation in this fungus and provide a theoretical basis for the high quality and high yield cultivation of this fungus.

蛹虫草是食药兼用且可以大规模培养子实体的真菌，具有较高营养价值和滋补活性。光照是其子实体发育和类胡萝卜素产生的关键因子，我们前期通过基因敲除发现光受体CmWC-1是其子实体发育和类胡萝卜素产生必不可少的一种转录因子，但是其调控机制不明。本项目以该转录因子为切入点，通过野生型和Cmwc-1敲除株光照前后及子实体发育不同阶段转录组比较分析，预测蛹虫草光受体CmWC-1调控的基因信息；染色质免疫共沉淀分析界定CmWC-1直接调控的靶基因及其结合的DNA序列特征；选择CmWC-1调控的重要转录因子及子实体发育和类胡萝卜素合成的相关基因，通过基因敲除及转录动态分析验证其功能，结合生物信息学分析，揭示CmWC-1、重要转录因子及子实体发育、类胡萝卜素合成相关基因之间的相互关系，最终阐明CmWC-1在子实体发育及类胡萝卜素产生中的调控机制，为蛹虫草子实体优质栽培和类胡萝卜素高效生产提供理论依据。

蛹虫草是食药兼用且可以大规模培养子实体的真菌，具有较高的营养价值和滋补活性。光照是其子实体发育和类胡萝卜素产生的关键因子，我们前期通过基因敲除发现光受体CmWC-1是其子实体发育和类胡萝卜素产生必不可少的一种转录因子，但是其调控机制不明。本项目以光受体CmWC-1为切入点，主要研究内容包括通过野生型和Cmwc-1敲除株光照前后及子实体发育不同阶段转录组比较分析，预测蛹虫草光受体CmWC-1调控的基因信息；染色质免疫共沉淀分析界定CmWC-1直接调控的靶基因及其结合的DNA序列特征；对CmWC-1直接调控的重要靶基因Cmvvd进行转录动态分析及基因敲除研究，揭示其在光信号传递及子实体发育、类胡萝卜素产生中的作用；通过对三种光受体CmVVD、CmWC-1和CmCRY-DASH之间的相互关系研究，揭示其在光信号传递及子实体发育、类胡萝卜素产生中的作用，从而阐明CmWC-1对子实体发育和类胡萝卜素合成的调控作用。本项目取得重要结果如下：.ChIP-seq分析得到CmWC-1调控的靶基因，基于靶基因启动子序列，生物信息学分析存在的共性基序（motif），得到CmWC-1 结合的DNA特征序列为AAATCAGRCCAC。ChIP-qPCR结果证实Cmvvd和Cmfrq为CmWC-1直接调控的靶基因。Cmvvd在原基阶段和幼嫩子实体阶段高表达，表明Cmvvd在子实体发育中可能起重要作用，基因敲除、回补和过表达试验证实ΔCmvvd中子实体发育受到明显抑制，Cmvvd对蛹虫草类胡萝卜素合成起负调控作用。阐明了三种光受体CmVVD、CmWC-1和CmCRY-DASH转录水平之间的相互关系，揭示Cmwc-1和Cmvvd、Cmwc-1和Cmcry-DASH在转录水平相互依赖，Cmcry-DASH表达受Cmvvd影响，Cmcry-DASH功能缺失可能会被高转录水平的Cmvvd补偿。在此基础上，提出了光受体调控蛹虫草子实体发育和代谢产物产生的模型。光受体感受光信号后，通过锌指蛋白类转录因子和G蛋白偶联的受体调控下游基因转录，从而调控子实体发育和代谢产物产生。本项目研究阐明光受体调控蛹虫草子实体发育的机制，为蛹虫草子实体优质栽培和类胡萝卜素高效生产提供理论依据。.项目发表第一作者或通讯作者英文论文11篇，中文核心期刊7篇，申请发明专利1项，培养硕博士研究生4名。