药用银杏叶中银杏酸合成的分子机制解析

银杏酸是一种长链烷基酚酸类代谢物，因其在食药类制剂中的毒性而被严格限制，故有效控制银杏酸合成是改良银杏叶品质的重要途径。目前关于银杏叶中银杏酸合成途径的分子机制尚未报道。我们前期选育了一株银杏酸含量显著降低的叶用银杏新品系，首先，我们发现具有相同碳链结构的银杏酸和脂肪酸的含量变化具有显著相关性；其次，利用序列比对筛选到了候选银杏酸合酶并找出了潜在的关键催化位点；定量分析发现它们的转录丰度与银杏酸含量呈显著正相关；最后，异源表达重组蛋白能催化脂肪酸合成相应的4-羟基银杏酸。据此，我们推测体内的银杏酸可能是由银杏酸合酶以脂肪酸为前体催化合成的。以此为切入点，本项目通过寻找银杏酸合成过程的主效基因和影响因子，鉴定银杏酸合成的限速酶并表征其催化活性，利用点突变验证其关键位点，以阐明银杏酸合成的分子机制，为有效控制银杏酸合成提供新思路和新靶点，进而为培育优质叶用银杏资源奠定理论基础。

Ginkgolic acids (GGA) is a family of natural 6-alkylresorcyclic acid metabolites, which alkyl chain usually has 13, 15, or 17 carbons and 0–2 double bonds like fatty acids (FA). Due to their negative attributes, the Chinese Pharmacopoeia limited at 10 μg/g the maximal permitted concentration of GGA in commercial phytopreparations, even a limit of 5 μg/g in the European and U.S. pharmacopoeias. The study on GGA mainly focused on preparation/removal, detection/quantification, chemical synthesis until now. However, the mechanism of biosynthesis in vivo is still unclear. We harvested a new Ginkgo biloba (Gb) strain characterized by lower ginkgolic acid content. FA which possess the similar carbon chain structure with GGA were all decreased significantly compared with the control through integrated analysis of metabolomics and transcriptomics. On the other hand, these FA were negatively correlated with GGA content at different growth stages of leaves. Two ginkgolic acid synthases (GGAS) were screened out and the key catalytic sites of them were identified via sequence alignment with other homologous gene. Their expression levels were positively correlated with GGA content. Finally, the recombinant proteins were available in vitro to produce 4-hydroxy-GGAS from the FA. Referring to previous studies on alkylresorcyclic acid synthase and our data, we speculated that ginkgo biloba translates the ginkgolic acid synthase (GGAS), which uses fatty acids as starter substrates to produce ginkgolic acids. In this project, we will monitor the content changes of GGA and FA during the growth stages to determine the metabolic correlation between them. Gene expression variation in samples with distinct metabolites differences mentioned above will be compared to find out the key genes and transcription factors involved in GGA biosynthesis. The GGAS will be characterized in vitro via heterologous expression. Finally, the key amino acid residual sites of GGAS involved in substrate specifity and carbon chain preference will be verified by site mutation. Consequently, we will elucidate the molecular mechanism of GGA biosynthesis by GbGGAS in the leaves of Gb. This study will provide new target and perspectives for GGA biosynthesis in Gb, which will contribute to breed a better germplasm of Ginkgo biloba.