蛹虫草麦角硫因及其生物合成基因的功能和作用机制研究

Aiming at the issue that Cordyceps militaris strain spawn degenerated commonly and seriously, based on our previous research results that strain spawn degeneration of Cordyceps militaris were closely related to oxidative stress and the contents of endogenous ergothioneine, and based on our results that there were two genes for ergothioneine biosynthesis in Cordyceps militaris genome, this project will over-express and knockout each individual gene and combinations of the two genes in ergothioneine biosynthesis of Cordyceps militaris by adopting international current leading-edge technology of CRISPR-Cas9. A series of mutation strains containing different expression content of ergothioneine will be created by over-expressing and knock-outing the each individual gene and combinations of the two genes through the genome editing technology of CRISPR-Cas9. Performance and their kinetic regularities of the mutation strains containing different expression content of ergothioneine will be detected and analyzed in strain spawn degeneration, growth and development, physiological and biochemical properties, other gene expressions, and their relationships. Then the effects of each individual gene, the combinatorial effects and the interaction effects of two genes will be determined and revealed. Combined with their functional characteristics of two genes through heterogenous expression and in vitro detection of their products, the function and their action mechanisms of ergothioneine and their biosynthetic genes in Cordyceps militaris will be elucidated by the comprehensive analysis of the individual gene effects, the combinatorial effects and their interaction effects of two genes, their functional characteristics in heterogenous expression and expression products in vitro. This research will expound the scientific principles of ergothioneine biosynthesis in Cordyceps militaris even in other edible fungi. And this research will obtain the genes of interests, regulatory elements, molecular markers, mutants and mutation strains of Cordyceps militaris with different content of ergothioeine and anti-degeneration. This will provide the genetic materials for the breeding of new outstanding varieties of Cordyceps militaris and other edible fungi with high content of ergothioeine and anti-degeneration. And this will provide theoretical guidance and technical supports for the development of novel technology in prevention and control of strain spawn degeneration by early detection, the regulation of cultivation and fermentation, etc. in Cordyceps militaris and other edible fungi.

本项目针对蛹虫草菌种退化普遍且严重的问题，基于前期研究发现蛹虫草菌种退化与氧化胁迫和内源麦角硫因含量密切相关、蛹虫草基因组中含有麦角硫因生物合成的两个基因的结果，计划采用国际前沿的CRISPR-Cas9基因组编辑技术对蛹虫草麦角硫因生物合成的两个基因进行单基因过量表达和双基因组合过量表达、单基因敲除和双基因组合敲除，创制蛹虫草单基因过表达菌株和双基因组合过表达菌株，创制蛹虫草单基因敲除菌株和双基因组合敲除菌株等麦角硫因不同表达量菌株，检测分析麦角硫因不同表达量菌株在菌种退化和生长发育、生理生化和其它基因表达等方面的表现及其变化规律，从而揭示单基因效应、双基因组合效应及其互作效应；结合异源表达和体外检测基因表达产物的功能特性，解析蛹虫草麦角硫因及其生物合成基因的功能和作用机制。为培育蛹虫草抗退化优异菌株提供遗传材料，为防控蛹虫草菌种退化提供早期检测和栽培管理的科学指导和技术支撑。

本项目的研究背景是：蛹虫草菌种退化普遍且严重，菌种退化与内源麦角硫因含量相关、蛹虫草基因组中含有麦角硫因生物合成基因同源序列。采用CRISPR-Cas9基因组编辑技术可以解析蛹虫草麦角硫因及其生物合成基因的功能特性和作用机制。. 本项目的主要研究内容和重要结果及关键数据有：完善了蛹虫草的CRISPR-Cas9基因组编辑技术，使得蛹虫草CRISPR-Cas9基因组编辑的成功率从原来的25%提高到94%，使得脱靶率从原来的31%降低到3%，即编辑精准率达到97%。建立了蛹虫草的Split-Marker基因组编辑技术，可作为蛹虫草基因组编辑的补充。蛹虫草麦角硫因的生物合成基因有两个，CmEgt1和CmEgt2；这与大多数食用菌和粗糙脉孢菌、米曲霉、粟酒裂殖酵母相同，都是两个基因；而与金针菇是3个基因、细菌是5个基因不同。蛹虫草的CmEgt1基因的核苷酸序列与其它食用菌的对应基因核苷酸序列的同源性为22.37%-59.66%，氨基酸序列与其它食用菌的对应序列的同源性为10.12%-56.28%，同源性都比较低，可视为新基因；CmEgt2基因的核苷酸序列与其它食用菌的对应基因核苷酸序列的同源性为24.24%-56.14%，氨基酸序列与其它食用菌的对应序列的同源性为15.69%-46.41%，同源性也都比较低，也可视为新基因。CmEgt1和CmEgt2的单基因过表达或双基因过表达都会提高蛹虫草内源麦角硫因的含量，而敲除任一基因都导致蛹虫草不能合成麦角硫因。蛹虫草的CmEgt1基因具有两个功能结构域，其在麦角硫因生物合成上的基因效应是CmEgt2基因的2.72倍。蛹虫草麦角硫因生物合成基因的缺失或突变及其导致的内源麦角硫因含量降低，是蛹虫草菌种退化的关键因素。. 本项目的科学意义：阐明了蛹虫草麦角硫因及其生物合成基因在蛹虫草菌种退化方面的功能和作用机制，为解决蛹虫草菌种退化问题奠定了科学基础。