蛹虫草隐花色素Cry-DASH功能及作用机制

Cryptochromes are blue-light/UV-A photoreceptors found in numberous organisms, which mediate photomorphogenesis and the regulation of circadian clock, and have been widely studied in plant and animal model organisms. Although the photoreceptor CmWC-1 has been demonstrated to sense light as a signal and transmit to cryptochrome Cry-DASH to modulate fruit body development and cordycepin production in Cordyceps militaris in our previous research, it was unillustrated that the function and mechanism of Cry-DASH in fruiting body development and cordycepin production of Cordyceps militaris. In this proposal, we will use a combination of approaches from structural biology, biochemistry and molecular biology to elucidate the function and molecular mechanism of cryptochrome Cry-DASH in cordyceps militaris. Special emphasis will be placed on 1) the knock-out of cry-DASH and alteration analysis of phenotype about the mutant strain will be performed to investigate the roles of Cry-DASH in the regulation of fungal fruiting body development and cordycepin production, 2) The tandem affinity puriftcation /MS is performed to identify proteins, which play key roles in the fruiting body development and cordycepin production, interacting directly with Cry-DASH, 3) the crystal structure of Cry-DASH is determined to analysis the catalytic mechanism, and generating the function of cryptochrome Cry-DASH in light reaction mechanisms with the resulting information and provide a theoretical basis for the high quality and high cordycepin yield cultivation of Cordyceps militaris.

隐花色素是广泛存在于生物体内的一类蓝光/UV-A受体，参与光介导的光形态建成及生物钟调控，在动植物模式生物中被广泛研究。前期研究证明蛹虫草光受体CmWC-1感受光信号后传递给隐花色素Cry-DASH，调控子实体发育和虫草素产量，但Cry-DASH调控蛹虫草子实体发育和虫草素合成的分子机制仍不清楚。本项目中，我们将综合利用分子生物学、结构生物学及生物化学研究手段对蛹虫草隐花色素Cry-DASH的功能和作用机制进行系统探索：通过隐花色素Cry-DASH基因敲除及表型分析，明确其对蛹虫草子实体发育和虫草素合成的作用；采用串联亲和纯化/质谱，鉴定与Cry-DASH直接相互作用并与子实体发育和虫草素合成相关蛋白；通过解析隐花色素Cry-DASH蛋白晶体结构，分析其催化机制，最终利用所得到的结果分析隐花色素Cry-DASH在光信号传递中的作用，为蛹虫草子实体优质栽培和提高虫草素产量提供理论依据。

光是蛹虫草子实体发育的关键因子，前期研究证实蛹虫草光受体CmWC-1感受光信号后传递给隐花色素CmCRY-DASH，调控子实体发育和虫草素产量。本项目以隐花色素CmCRY-DASH为切入点，主要研究内容包括通过Cmcry-DASH转录动态分析及基因敲除研究，明确其在光信号传递及子实体发育、虫草素产生中的作用；采用串联亲和纯化/质谱，鉴定与CmCRY-DASH直接相互作用的蛋白；串联表达纯化法验证与CmCRY-DASH直接相互作用的蛋白；对CmCRY-DASH调控的光受体基因Cmvvd的转录动态分析和基因敲除研究，解析CmCRY-DASH对蛹虫草生长发育和虫草素合成的调控机制。.本项目取得重要结果如下：.Cmcry-DASH基因表达受光照强烈诱导，且依赖于CmWC-1；转录动态、基因敲除、回补和过表达试验证实Cmcry-DASH调控子实体生长发育，并负调控虫草素的合成；两种光受体基因Cmwc-1和Cmcry-DASH在转录水平互相依赖。坐滴法筛选到CmCRY-DASH蛋白晶体。采用TAP/MS法鉴定到与CmCRY-DASH直接相互作用的蛋白71个，同时采用串联表达纯化法验证了CmCRY-DASH与热休克蛋白HSP70（CCM_04804）之间的直接相互作用，且与野生型相比较，△Cmcry-DASH失去对热激的响应能力，因此推测CmCRY-DASH参与蛹虫草对环境因子-温度的响应；Cmcry-DASH 敲除株中Cmvvd见光后表达量大幅升高以补偿Cmcry-DASH的缺失，Cmvvd同源重组敲除实验证实Cmvvd调控蛹虫草子实体发育，并对蛹虫草虫草素合成起正调控作用。本项目研究阐明了光受体CmCRY-DASH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虫草素高效生产提供了理论依据。.项目发表第一作者或通讯作者SCI论文4篇，非通讯非第一作者SCI论文4篇，其他论文4篇，会议摘要9篇，申请发明专利1项，参与指导4名硕博士研究生的课题研究，并负责指导1名本科生完成毕业设计。