胡萝卜素β-和ε-环羟化酶相互作用协同调控发芽玉米富集叶黄素的机制

Lutein can protect against age-related macular degeneration. Carotene hydroxylases play a critical role in lutein biosynthesis. It catalyzes the hydroxylation of β- and ε-rings of α-carotene for the synthesis of lutein. Preliminary studies found that the expression of carotene hydroxylase genes were significantly influential on lutein accumulation. However, the roles of carotene hydroxylases in regulating lutein accumulation in the germinated corn kernels, and a synergistic interaction between carotene hydroxylases are still unclear. This project will study the effects of β- and ε-hydroxylase activity, gene and protein expression levels on the accumulation of lutein in the germinated corn kernels. Meanwhile, the roles of β- and ε-hydroxylase and their synergistic effects on lutein synthesis were studied by Escherichia coli functional complementation system. And then, the interactions between β-hydroxylase and ε-hydroxylase proteins were demonstrated by protein interaction assays. Further, the regulation of β- and ε-hydroxylase on lutein enrichment was examined by their inhibitor treatments. Finally, the synergistic interaction mechanism of β- and ε-hydroxylase on the enrichment of lutein in germinated corn could be clarified. This project will not only enrich the scientific theory of xanthophylls synthesis by carotenoid hydroxylases, but also provide technological support for heterologous synthesis of lutein.

叶黄素能够预防老年黄斑变性，胡萝卜素羟化酶是叶黄素生物合成的关键酶，催化α-胡萝卜素的β-和ε-环羟基化后生成叶黄素。申请人前期研究发现玉米籽粒发芽后叶黄素含量显著提高，且与β-和ε-环羟化酶基因表达显著相关，但β-环与ε-环羟化酶两者之间的协同调控作用机制尚不明确。本项目将研究发芽玉米不同生长阶段中β-和ε-环羟化酶活性、基因和蛋白表达量变化规律及对叶黄素含量累积的影响；以大肠杆菌功能表达系统为平台，探究β-和ε-环羟化酶对叶黄素合成的作用及协同效应；进一步通过蛋白互作试验，揭示多酶复合物中β-环与ε-环羟化酶蛋白之间的相互作用模式，结合β-和ε-环羟化酶抑制处理，解析β-和ε-环羟化酶对叶黄素富集的调控作用，系统阐明胡萝卜素β-和ε-环羟化酶相互作用协同调控发芽玉米富集叶黄素的机制。该研究将丰富胡萝卜素羟化酶合成叶黄素类物质的科学理论，为胡萝卜素羟化酶异源合成叶黄素类物质提供技术支撑。

叶黄素具有能够预防老年黄斑变性等重要的生理功能，而胡萝卜素羟化酶是植物体内叶黄素生物合成的关键酶，催化α-胡萝卜素的β-和ε-环羟基化后生成叶黄素。研究结果表明，发芽过程可显著提高玉米籽粒中类胡萝卜素的含量，其中叶黄素和玉米黄质含量提高约为2~3倍；胡萝卜素羟化酶基因表达量与叶黄素含量的累积呈显著相关；外源胁迫条件可通过上调或下调番茄红素环化酶及胡萝卜素羟化酶基因表达量，从而促进发芽玉米中叶黄素和玉米黄质的合成；本项目成功克隆玉米胡萝卜素羟化酶CYP97A、CYP97B、CYP97C、BCH1和BCH2基因，并成功在大肠杆菌B21(DE3)中表达；根据高效液相色谱产物分析推断，胡萝卜素羟化酶CYP97A和CYP97C分别具有β-环和ε-环羟化酶活性，而CYP97B具有β-环羟化酶活性；双分子荧光互补试验证实胡萝卜素羟化酶CYP97A、CYP97B、CYP97C蛋白之间存在相互作用。因此，胡萝卜素羟化酶蛋白的互作，会形成代谢单元，使得代谢底物在空间上接近，有效地催化α-胡萝卜素生成叶黄素。本项目解析了发芽玉米中类胡萝卜素合成酶对叶黄素合成的介导作用，并通过大肠杆菌体外表达系统初步探明胡萝卜素羟化酶的功能及胡萝卜素羟化酶相互作用协同调控叶黄素合成的机制，为生物合成叶黄素类物质提供科学理论和技术支撑。