VeA蛋白协同调控灵芝有性发育与三萜类成分积累的机制研究

Ganoderma lucidum involves multiple biosynthetic pathways of secondary metabolites, making it an ideal model to study the biosynthesis of secondary metabolites and their regulations. Recently, the elucidation of the chromosome-level genome has laid the solid foundation for further application of G. lucidum as a medicinal model organism. G.lucidum is supposed to play important roles in the study on the diversity of secondary metabolites, development of medicinal fungi and synthetic biology of natural medicines. The analysis of G.lucidum’s genome implicated that VeA maybe play a critical role in the coordination of G.lucidum sexual development and terpenoid accumulation. VeA is one of the most widely studied protein in fungal secondary metabolism and development regulation. VeA exerts its function through the velvet complex in vivo while VeA/VelB/LaeA heterotrimer stands at the key role of this complex. The formation of velvet complex is dependent on light signal pathway.The components of G.lucidum velvet complex and it’s regulation network are still unknown.In this study, we will use Co-Immunoprecipitation, yeast two-hybrid system, RNA interference, Gene Overexpression，complement experiment and RNA-seq to investigate the constitution of G.lucidum velvet complex and the regulation network of VeA in G.lucdum.This work will enhance the model status of G.lucidum in traditional medicine research as well as pave the way for G.lucidum cultivation improvement and breeding.

灵芝（Ganoderma lucidum）是研究发育与次生代谢协同调控的中药基原模式生物，申请人通过灵芝的全基因组分析发现灵芝VeA蛋白与灵芝有性发育和三萜类成分积累相关联[Nature Communications，2012]。VeA蛋白是目前研究最为深入的真菌次生代谢与发育协同调控蛋白。在细胞核内，VeA与VelB及LaeA形成velvet蛋白复合体的核心三聚体。velvet复合体的组成决定次生代谢产物的全局调控。本研究拟通过免疫共沉淀，酵母双杂交，RNA干扰，基因过表达，互补实验和转录组测序等分子生物学技术从内源和外源两个方面确定VeA在灵芝中的互作蛋白，velvet复合体的组成及其对三萜类物质的调控网络，系统阐释灵芝中发育与次生代谢协同调控的分子机制，进一步加强灵芝的药用模式真菌作用，并为以灵芝酸生产为目的的灵芝栽培技术的改进以及灵芝品种的选育提供理论依据。

本研究以药用模式生物灵芝为平台，系统研究了灵芝中Velvet家族蛋白并克隆GlVeA基因。定位并分析灵芝交配型位点A和B，解释了灵芝在发育过程中四级异宗特性，通过转录组发现费洛蒙及其受体与Velvet家族蛋白有关联。完成紫芝全基因组测序，表明velvet蛋白家族在灵芝属中保守。在调控机制上，通过二倍体重测序、甲基化测序和转录组分析发现灵芝Velvet家族蛋白编码基因，发育相关基因及次生代谢表达基因不仅受到转录层面的调控，而且经历翻译后修饰和5mC导致的点突变的表观遗传调控。在灵芝属全局比较过程中，我们完成了一套基于光学图谱的快速比较基因组研究方法，同时开发了一套快速鉴定灵芝属交配型的引物。本研究将为灵芝栽培技术的改进和遗传育种提供基础。