光照对三孢布拉霉中类胡萝卜素合成的诱导效应及其机制研究

Due to the ability to synthesize carotenoids that has important use, Blakeslea trispora has received intense attentions. The synthesis of carotenoids in B. trispora is induced by light. This phenomenon is much rarely studied in B. trispora, which limits the synthesis level of carotenoids. This project aims to study the effect and its mechanism in molecular genetics, physiology, multi-level regulation: (1) to explore effects of different light wavelengths, doses and illumination modes on the formation of carotenoids; (2) In view of the light receptor protein WC-1 and repressor protein CrgA, to analyze of the effect of light induction on structure gene expression and carotenoid synthesis by using high efficient gene knockout or overexpression technologies; (3) to investigate effects of quenching of endogenous reactive oxidative radicals and addition of exogenous reactive oxidative radicals and response of intracellular antioxidant enzyme and carotene synthesis, which helps us to analyze the association between light induction effect and oxidative stress; (4) to analyze synthesis kinetics of mRNA level of carotenoid structure gene, enzyme level and carotenoid content level, to add inhibitors of gene transcription and translation and to analyze enzymatic activities associated with carotenoid structure genes, for integral clarification of mechanism of carotenoid synthesis by light induction at milti-level regualation. Launch of this project will provide a theoretical basis to reveal the effect and mechanism of carotenoid synthesis by light induction in B. trispora.

三孢布拉霉能合成具有重要用途的类胡萝卜素而广受关注，其生物合成受光诱导调控，但因对该效应研究较少而制约了其合成水平。本项目拟研究这一效应并从分子遗传学、生理学和多水平调控等角度阐述其机制：（1）研究光不同波长、剂量和照射模式对类胡萝卜素合成的影响；（2）针对光受体蛋白WC-1和阻遏蛋白CrgA，采用高效基因敲除技术或过表达技术，分析其对光诱导类胡萝卜素结构基因表达和类胡萝卜素合成的影响；（3）通过猝灭内源活性氧、添加外源活性氧试验以及抗氧化酶和类胡萝卜素合成的响应研究光诱导效应与氧胁迫的关联性；（4）构建不同条件下类胡萝卜素结构基因mRNA水平、酶活水平和类胡萝卜素合成水平动力学，另外进行基因转录、翻译抑制试验和类胡萝卜素结构基因酶系的酶活和类胡萝卜素含量测定，综合分析光对类胡萝卜素合成的多水平调控机制。本项目的开展将会为揭示三孢布拉霉中类胡萝卜素合成的光诱导效应及机制提供一定的理论依据。

三孢布拉霉能合成具有重要用途的类胡萝卜素而广受关注，其生物合成受光诱导调控，但因对该效应研究较少而制约了其合成水平。本项目开展了光诱导类胡萝卜素合成的研究并从分子遗传学、生理学和多水平调控等角度试图阐述其调控机制。项目从以下几个方面开展：（1）研究了光不同波长、剂量和照射模式对类胡萝卜素合成的影响，显示蓝光和白光对类胡萝卜素合成具有显著诱导效应，且短时间、高强度光照效果最好，长时间光照可能引起类胡萝卜素降解从而降低产量；（2）建立了巢式PCR方法克隆获得了光受体蛋白BTWC-1（A和C），生物信息学分析表明它们与瓜笄霉和卷枝毛霉相似度最高，具有光受体特征的LOV结构域，蓝光和白光照射显著提升btwc-1c和类胡萝卜素结构基因carB的转录。利用卷枝毛霉muwc-1a和muwc-1c功能缺失突变株对BTWC-1A和BTWC-1C进行的体内功能分析表明他们分别与调控菌丝体趋光性和类胡萝卜素合成相关。克隆获得了CrgA，基因敲除和回补研究表明其能控制类胡萝卜素合成、产孢水平和胞内抗氧化酶水平，其调控类胡萝卜素合成机制在于其对类胡萝卜素结构基因转录水平调控、酶活水平调控以及代谢水平影响。光照同时能影响CrgA对孢子生成和类胡萝卜素合成的调控。（3）光照诱导活性氧生成和类胡萝卜素合成相关性研究表明，短时间光照能产生大量ROS，并诱导类胡萝卜素合成，通过猝灭内源活性氧、添加外源活性氧研究揭示光诱导效应可能受ROS介导，并在基因转录水平发挥作用。（4）进行了类胡萝卜素光诱导合成多水平调控分析，结果表明光照可能在转录水平和酶水平调控类胡萝卜素合成。本项目的开展将会为揭示三孢布拉霉中类胡萝卜素合成的光诱导效应及机制提供一定的理论依据。