黄芩中黄芩素、汉黄芩素合成途径中黄酮羟化酶的研究

Scutellaria baicalensis Georgi is an important Chinese medicinal plant. Baicalein, wogonin and their glucosides are the bioactive compounds in Scutellaria. To date, people have elucidated the metabolic pathway from phenylalanine to chrysin. However, it is unclear how chrysin is decorated to produce baicalein and wogonin, the whole pathway remains to be elucidated. In theory, chrysin may be 6-hydroxylated to produce baicalein and 8-hydroxylated and 8-methyltrasferation to produce wogonin, respectively. Based on previous work, we have analysed RNA sequencing database from root and flower tissues of Scutellaria. Combined with q-PCR study, we have obtained several candidate genes may be responsible for hydroxylation of chrysin. In this study, our group plan to assay the enzyme activity using yeast in vivo system, study their kinetics to find their natural substrates. We will confirm contributions of the enzymes to the pathway through hairy root mediated RNAi, detect their functions and activities in planta through sub-cell localization and transgenic Arabidopsis. Finally, we will reconstruct the pathway using tobacco leaf infiltration. The results of this present work could be necessary to elucidate the whole biosynthesis pathway of baicalein and wogonin.

黄芩是一种重要的中国药用植物，黄芩素、汉黄芩素及其糖苷为黄芩中主要活性成分。目前的研究已阐明了从苯丙氨酸到白杨素的合成途径。然而，还不清楚是哪些酶参与从白杨素到黄芩素、汉黄芩素的合成。理论分析，白杨素分别需经过6位羟化形成黄芩素；经过8位羟化及8位氧甲基化形成汉黄芩素。在前期工作中，申请者分析了黄芩根、花的转录组数据，结合qPCR表达特征，筛选到了一些黄酮羟化酶候选基因。本项目拟通过酵母体内酶活检测体系检验这些候选基因的活性，通过酶促反应动力学实验来测定这些酶的动力学常数，判断它们的天然底物。用发根介导的RNA干扰来检测它们对于整个代谢途径的贡献。通过亚细胞定位及转基因拟南芥来验证它们在植物细胞内的活性，最后用烟草细胞瞬时表达体系来重构这些已阐述的合成途径。本项目的实施，将为整个黄芩素、汉黄芩素合成途径解析提供重要环节。

黄芩是一种重要的中国药用植物，黄芩素、汉黄芩素为黄芩中主要活性成分。黄芩素，汉黄芩素应该由白杨素经过6位或8位羟基化及甲氧基化合成而来。我们分离和验证了黄芩中两个CYP450家族酶， SbCYP82D1.1及SbCYP82D2。生化实验表明，它们分别为黄芩中的黄酮6-羟化酶（F6H）和黄酮8-羟化酶（F8H）。SbCYP82D1.1对黄酮类如白杨素和芹菜素具有广泛的底物接受性，负责根中的黄芩素和地上部分的野黄芩素的合成。当用RNAi技术在黄芩毛状根中降低SbCYP82D1.1的表达时，黄芩苷和黄芩素水平显著降低，而白杨素苷则在毛状根中积累。 CYP82D2是一种具有底物特异性较高的F8H，只接受白杨素为底物，产生去甲基汉黄芩素。系统发育分析表明，CYP82D2可能通过基因复制从SbCYP82D1.1进化而来，因而保留了部分祖先酶F6H的活性。本工作找到了黄芩素，汉黄芩素合成途径中的羟化酶，阐明了黄芩素、去甲汉黄芩素的完整合成途径，完成了本项目计划的目标和任务。发表论文三篇（其中SCI论文两篇），申请专利两件。这些工作为黄芩分子育种及用合成生物学生产这些黄酮物质提供了理论基础及工具。