基于LiP/iTRAQ靶蛋白鉴定技术解析HSYA生物合成途径羟化酶的功能机制

红花主要活性成分是含有查耳酮母环的羟基红花黄色素A，广泛用于预防和治疗心脑血管疾病，下游合成途径不明且来源单一是限制其药源供应和可持续开发的重要原因。羟基化末端修饰的多样性是解析途径的难点。前期预测查耳酮母环通过CYP450合成HSYA前体，并通过转录组筛选到10条CYP450，进而通过分子对接和“基因-化合物”相关分析筛选到候选CYP450潜在参与HSYA生物合成。基于此，本项目拟采用瞬时表达和异源诱导方法探析候选基因亚细胞定位及构象特性；利用酵母微粒体模块快速验证候选基因体外催化活性；通过CRISPR/Cas9技术编辑红花植株，开展LiP/iTRAQ靶蛋白筛选，精准鉴定HSYA结合CYP450靶蛋白结合位点。本项目旨在探索CYP450参与调控HSYA生物合成功能机制,以解析HSYA生物合成途径为目标，为HSYA异源全合成奠定基础。

Hydroxysafflor Yellow A (HSYA), uniquely existing in floret in Carthamus tinctorius L., is an active compound to prevent and treat cardiovascular & cerebrovascular diseases. Unclear biosynthetic pathway and limited wild resource are the main reasons that limiting supply and sustainable exploitation. Diversity of hydroxylases is the major sticking point for pathway analysis. Previous study has predicted chalcone precursor of HSYA would be catalyzed by CYP450. Through the transcriptome analysis, 10 candidate CYP450s were screened out. Following by docking analysis and “gene-compound” pearson correlation, candidate CYP450 was identified to potentially involve in HSYA biosynthetic pathway. Based on the previous results, the project will be aimed to explore subcellular location and protein confirmation of candidate CYP450 by transient expression and IPTG-induction. Then yeast microsomal system will be used to validate the enzymatic activity. Finally, CRISPR/Cas9 gene editing system and LiP/iTRAQ will be used to identify protein targets combining with HSYA accurately. Overall, this project intends to explore functional mechanism of CYP450 regulating HSYA and detailed HSYA biosynthetic pathway which will provide the foundation for heterogeneous synthesis of HSYA.