项目名称: 新疆紫草中紫草素生物合成途径关键基因GHQH的克隆及功能研究
项目编号: No.81473307
项目类型: 面上项目
立项/批准年度: 2015
项目学科: 医药、卫生
项目作者: 郭兰萍
作者单位: 中国中医科学院中药研究所
项目金额: 75万元
中文摘要: 紫草素类化合物是新疆紫草中的主要有效成分,其具有多种药理作用和巨大经济价值。香叶烯醌-3'-羟化酶(GHQH)是植物中发现的第一个具有这一修饰功能的羟化酶,是紫草素生物合成途径的关键酶。研究组前期筛选获得新疆紫草紫草素类高产红色系和低产白色系,本研究以红色系和白色系的代谢谱和转录表达谱差异分析为基础,筛选获得紫草素生物合成途径关键酶GHQH基因,并构建酵母工程菌株,获得大量紫草素生物合成过程中GHQH催化底物GBA,通过真核表达、体外酶促和全细胞催化等对筛选获得的GHQH基因进行功能验证。同时从GHQH基因过表达和表达抑制正反两个角度,阐明其在紫草素类形成中的作用。本研究综合化学、分子生物学、生物信息学和合成生物学的方法,进行紫草素生物合成关键酶基因GHQH的克隆和功能分析,可为深入解析紫草素生物合成途径,通过基因表达调控提高紫草素产量提供理论依据,对缓解新疆紫草野生资源的压力有重要意义。
中文关键词: 新疆紫草;紫草素;生物合成;功能基因;GHQH
英文摘要: Shikonins are the main active ingredients in Arnebia euchroma, which have a variety of pharmacological effects and great economic value. Geranylhydroquinone 3''-hydroxylase(GHQH), the first enzyme of this modification function found in plants ,is one of key enzymes in shikonin biosynthesis. We gained two cell lines of A. euchroma: the shikonin proficiency red cell line and shikonin deficiency white cell line in previous study. Based on the differences analysis of metabolic profiles and transcriptional expression profiles of red line and white line, we screened and gained the predicted genes encoding GHQH in shikonin biosynthesis. In this program we are going to construct a yeast engineering strain to produce GBA, the substrate of GHQH, and then through, enzymatic reaction in vitro, and whole-cell catalysis to verify the function of predicted GHQH. Meanwhile, in vivo, we will use RNA overexpression and RNA interference of GHQH to clarify the role of GHQH in shikonin biosynthesis. In this study, we intent to clone and analysis the function of gene encoding the key enzyme GHQH in shikonin biosynthesis of A. euchroma by a comprehensive chemistry, molecular biology, bioinformatics and synthetic biology method, and this work will provide a theoretical basis for improving shikonin yields by regulation of gene expression and may help to relief the stress of the wild A. euchroma resources.
英文关键词: Arnebia euchroma;shikonin;biosynthesis;functional-gene;GHQH