黑果枸杞果实中主要花青苷的高效合成机制研究

特色药食同源物种黑果枸杞（Lycium ruthenicum）果实中最主要的活性成分是花青苷，其具有治疗阿尔兹海默症等疾病的潜力，但黑果枸杞花青苷合成途径特别是下游具有结构稳定作用的糖基化和酰基化修饰尚不清楚。本项目前期获得了黑果枸杞花青苷的糖基转移酶和酰基转移酶候选基因，在初步测定了重组蛋白活性的基础上，进一步用大肠杆菌体系研究候选基因的体外功能、通过酶的特异性确定修饰酶的催化顺序，并采用烟草瞬时表达技术验证体内功能，结合不同组织和果实不同发育时期的基因相对表达水平，解析黑果枸杞果实中主要花青苷的修饰途径；在此基础上，利用BiFC和Y2H技术探索候选蛋白之间的互作关系，建立修饰酶形成的代谢区室模型，进而阐明黑果枸杞果实中不稳定花青素到稳定花青苷高效合成的分子机制。本项目的研究结果将为黑果枸杞的分子育种提供有效的基因靶点，亦为花青苷的生物合成提供基因资源和理论指导。

Anthocyanins are the main active components in black goji (Lycium ruthenicum), a well-known medicinal and edible plant in China. The stability of anthocyanins are attributed to various glycosylated and acylated modifications. However, the modification pathway of anthocyanin in black goji is still unclear. In our previous study, several candidate glycosyltransferase and acyltransferase genes from L. ruthenicum were cloned, also the catalytic activity of the recombinant enzymes was tested. Thus, this project will use E.coli expression system to fully characterize the in-vitro function of these candidate genes, and the modification order of candidate enzymes will be clarified with enzyme specificity. Tobacco transient expression will be used to elucidate the in-vivo function. Then the relative gene expression level in different tissue or different developmental stages of fruits will be quantified using qRT-PCR. Furthermore, we will identify protein-protein interaction between those modification enzymes and build a model of metabolon using bimolecular fluorescent complementation (BiFC) system and yeast two-hybrid (Y2H) assay. The metabolon with multiple enzymes will be explored for high-efficiency modification process of the anthocyanin. Overall, this project will focus on the mechanism of high efficiency synthesis of anthocyanin in L. ruthenicum. The results will provide target genes for molecular breeding of new cultivars and metabolic engineering of anthocyanins.