环境盐胁迫触发北冬虫夏草新型类胡萝卜素高度累积的分子调控机制

The edible and medicinal fungi Cordyceps militaris that contains many different kinds of physiologically active compounds has similar active ingredients composition and pharmacological effects to those of the famous edible and medical fungus Cordyceps sinensis. Previous studies revealed that C. militaris had the ability to accumulate the novel bioactive carotenoids. However, the biosynthetic pathway has not been fully elucidated. Moreover, it was found that the accumulation of carotenoids in C. militaris significantly increased under salt stress conditions. Based on the scientific understandings of carotenoid biosynthetic pathway and its molecular regulation mechanisms in fungi, this project will isolate and characterize the carotenogenic genes of C. militaris using technologies on comparative transcriptome and molecular cloning. The actual catalytic function and efficacy of speculative carotenogenic enzymes will be investigated by the methods of functional characterization of complementary genes, mass spectrometric analysis and chromatographic analysis. As a result, the biosynthetic metabolic network of novel carotenoids in C. militaris will be study in this work. In addition, the genetic engineering strains with diverse products will be constructed with different combinations of the carotenogenic genes from C. militaris followed by the optimization for high carotenoid accumulation. Subsequently, triacylglycerol biosynthesis and mitogen-activated protein (MAP) kinase pathway responsible for the salt stress condition will be investigated using techniques of fluorogenic quantitative PCR, and Western Blot assay/enzyme-linked immunosorbent assay (ELISA). Changes of expression profiles of carotenogenic genes will be further study on MAP kinases (Slt2 and Hog1) gene knockout strains constructed by Cre/loxP recombination system under the salt stress conditions. Finally, the molecular regulatory mechanism underlying high carotenoids accumulation of C. militaris triggered by salt stress factor will be displayed based on the studies above. This research will lay a scientific foundation and provide technical assistance for the understanding of molecular regulation of carotenogenesis and the increased accumulation of novel bioactive carotenoids in C. militaris. In addition, this work will promote the production of these novel carotenoids by genetic engineering strains harboring carotenogenic genes of C. militaris.

北冬虫夏草含有多种生理活性化合物，具有与冬虫夏草相近的活性成分和药理作用。前期研究显示北冬虫夏草可合成新型高生物活性类胡萝卜素，但其生物合成路径尚未被阐明。我们发现环境盐胁迫压力能够显著提高北冬虫夏草的类胡萝卜素累积量。本项目将基于真菌类胡萝卜素生物合成代谢及其分子调控的科学认识，利用比较转录组、分子克隆、基因功能互补、质谱和色谱分析等技术获取北冬虫夏草类胡萝卜素合成酶基因并解析其生物学功能和效能，揭示其类胡萝卜素生物合成代谢网络，构建基于北冬虫夏草类胡萝卜素生物合成路径的产物多样性基因工程菌株并实现其高效表达。接着，结合荧光定量PCR、蛋白表达分析、基因敲除等手段，从脂质合成和MAPK级联通路两方面探讨北冬虫夏草响应盐胁迫因子触发类胡萝卜素高度累积的分子机理。本研究为深入解释和提高北冬虫夏草类胡萝卜素的生物合成以及利用基因工程菌株生产新型类胡萝卜素的实质应用奠定科学基础和提供技术支持。

北冬虫夏草含有多种生理活性化合物，具有与冬虫夏草相近的活性成分和药理作用。前期研究显示北冬虫夏草可合成新型高生物活性类胡萝卜素，但其生物合成路径尚未被阐明。项目研究基于真菌类胡萝卜素生物合成代谢及其分子调控的科学认识，利用比较转录组分析、分子克隆、基因功能互补、高效液相色谱分析等技术发掘了Cmcao-2、Cmylo-1、CmPsy、CCM_01235、CCM_4200、CCM_09369、easc、erg、ts、cat、hsp78、CmTns、CmFhp、CmMox和CmHyp等北冬虫夏草类胡萝卜素合成功能酶基因并解析其生物学功能和效能，揭示了其类胡萝卜素生物合成代谢网络，构建基于北冬虫夏草类胡萝卜素生物合成路径的产物多样性基因工程菌株。接着，结合荧光定量PCR、基因过表达和基因敲除等手段对功能酶基因进行解析，从类胡萝卜素合成代谢途径、MAPK信号通路、脂质合成代谢、热休克蛋白表达等四方面揭示盐胁迫等环境应激因子触发北冬虫夏草新型类胡萝卜素高度累积的分子调控机制。本研究为深入解释和提高北冬虫夏草类胡萝卜素的生物合成以及利用基因工程菌株生产新型类胡萝卜素的实质应用奠定科学基础和提供技术支持。