GPI锚定蛋白在香芹酚调控枸杞链格孢菌丝细胞壁构建中的作用机理研究

GPI锚定修饰介导的细胞壁构建是病原真菌抵抗不良环境以及逃避宿主免疫的重要策略。香芹酚可调控枸杞黑霉病菌菌丝细胞壁构建，但其对GPI锚定修饰的调节模式尚不明了。本项目以枸杞黑霉病菌为研究对象，通过DIA蛋白质组分析、生物信息学预测结合实时荧光定量PCR分析来研究香芹酚调控的GPI锚定蛋白类型。通过构建GPI锚定蛋白基因敲除突变体，利用透射电子显微镜、激光共聚焦显微镜和荧光显微镜等细胞生物学手段研究GPI锚定修饰调节对黑霉病菌的菌丝生长、细胞结构、细胞壁基质代谢、GPI锚定蛋白分布及香芹酚耐受性的影响，分析GPI锚定修饰结合药物处理对病菌与果实互作中病菌致病性和宿主免疫能力的作用规律。通过上述研究，在分子、生化和细胞水平解析香芹酚调节GPI锚定修饰在枸杞黑霉病控制中的功能作用，为解析GPI锚定修饰在链格孢菌致病中作用机制奠定基础，也为有效利用香芹酚及开发新型抗菌药物提供依据。

Cell wall architecture mediated by GPI anchored modification is recognized as an important strategy for pathogenetic fungal against adverse environment and escape host immunity. The cell wall construction of Alternaria alternata, which caused black mold in fresh fruit of Lycium barbarum, could be regulated by carvacrol. However, the mode of GPI anchored modification mediated by carvacrol has not been understood. In this project, the pathogens resulted in black mold of Goji berry are chosen as research object. The types of GPI anchored proteins regulated by carvacrol are elucidate by modern analyzed methods of DIA proteomics, bioinformatics combined with RT-PCR. Furthermore, GPI anchored protein gene knockout mutants are constructed in the further investigations. The effect of regulation of GPI anchored modification on mycelial growth, cell structure, metabolism of cell wall matrix, distribution of GPI anchored proteins and tolerance of carvacrol in different strains are observed by modern techniques of TEM, LSCM and fluorescence microscope. Meanwhile, the effect of GPI anchor modification combined with carvacrol treatment on pathogenicity of pathogens and host immunity in the interaction between pathogen and fruit are also investigated. Based on the above experiments, the functional roles of GPI anchored modification regulated by carvacrol on control of black mold of Goji berry will be clarified at molecular, biochemical and cell levels. The results will provide a theoretical basis for further understanding mechanism of GPI anchored modification on pathogenicity of A. alternata. It also provides the basis for the effective utilization of carvacrol and the development of new antifungal drugs.