光亲和标记荧光探针介导朝藿定C生物合成关键糖基转移酶的发现及功能研究

淫羊藿为我国传统补肾壮阳中药，朝藿定C是其主要药效成分之一，具有促进睾丸细胞增殖、改善男性生殖功能等药理作用。已有研究表明，朝藿定C的生物活性与其高度糖基化密切相关，但催化其前体化合物淫羊藿苷连接糖基生成朝藿定C的糖基转移酶及其糖基化过程仍未阐明，亟待解析。近年来光亲和标记荧光探针技术在钓取天然产物生物合成关键酶的研究中凸显精准、特异等优势。申请人前期从淫羊藿中制备了山萘酚和淫羊藿苷等前体化合物，并在解析山萘酚生物合成途径的基础上设计合成了山萘酚和淫羊藿苷的多种光亲和标记荧光探针。本项目拟通过光亲和标记荧光探针的设计和优化定向筛选朝藿定C糖基化途径中特有的糖基转移酶，并系统解析其结构和功能，阐明朝藿定C的糖基化后修饰过程。通过本项目的实施，为后续搭建植物基因异源表达体系以及生物合成更丰富、活性更显著的结构类似物奠定基础，并为中药活性天然产物生物合成关键酶的精准挖掘提供借鉴。

Epimedium brevicornum is a well-known traditional Chinese medicine and has good effects on tonifying kidney and strengthening Yang. Epmedin C is the effective constituent with pharmacological effects on promoting testicular cell proliferation and improving male reproductive function. .Recent reports indicated that glycosylation is closely related to the bioactivity of Epmedin C. However, there are few studies on glycosyltransferases related to catalyze Icariin to Epmedin C. Until now, the biosynthesis pathway of the Epmedin C remains unclear. In recent years, the photo-affinity labeled fluorescent probe technology has become more precise and specific for fishing key enzymes in biosynthesis of natural products. Previously, this project had prepared precursors such as kaempferol and Icariin from E. brevicornum, and designed and synthesized a variety of photo-affinity labeled fluorescent probes for precursors on the basis of elucidating the biosynthesis pathway of kaempferol..To further elucidate the complete biosynthesis pathway of Epmedin C, we will design several photo-affinity labeled fluorescent probes to find the exact glycosyltransferase involved in Epmedin C synthesis pathway and characterize the function of these glucosyltransferases. We believe that the implementation of this project will not only lay a foundation for diversifying the Epmedin C structure but also benefit its heterogeneous expression system construction. This project could also bring a light to key enzymes mining of plant natural products biosynthesis.