The synthesis of diterpenoid tanshinones, which are some of the main bioactive ingredients of Danshen (the roots of Salvia miltiorrhiza ), begins with the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy- d -xylulose-5-phosphate synthase (DXS) catalyzes the first step of the MEP pathway and is considered to be a key rate-limiting enzyme. To confirm the function of DXS genes, in this study two isoforms of DXS ( SmDXS1 and SmDXS2 ) were isolated by rapid amplification of DNA ends from leaves of S. miltiorrhiza. Bioinformatic analyzes indicated that two SmDXSs had high homology with other plant DXSs. Tissue expression pattern analysis revealed that SmDXS1 and SmDXS2 exhibited distinct expression patterns in S. miltiorrhiza. SmDXS1 was expressed in all analyzed tissues; the highest expression was observed in leaves, followed by stems, with weak expression in roots. In contrast, SmDXS2 transcripts were predominantly detected in roots, but very weakly. Subcellular localization analysis revealed that SmDXS1 and SmDXS2 were both localized in the chloroplast. Agrobacterium -mediated transformation was used for induction of transgenic hairy root lines and tanshinone yield was assessed by high-performance liquid chromatography. Compared with the control, overexpression of SmDXS1 and SmDXS2 significantly enhanced tanshinone accumulation in transgenic roots, whereas only down-regulation of SmDXS2 led to a significant decrease. Our results indicated that SmDXS2 was an important rate-limiting gene in the MEP pathway involved in tanshinone biosynthesis and could be an effective regulatory target for controlling tanshinone accumulation in S. miltiorrhiza hairy root cultures.

中文翻译：

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两个1-脱氧-的分子克隆和表征 d 在参与生物合成丹参酮-xylulose -5-磷酸合酶基因 丹参

二萜丹参酮，它们是一些丹参的主要生物活性成分（根部的合成 丹参 ），开始于质体甲基赤4-磷酸（MEP）途径。1-脱氧 d -xylulose-5-phosphate synthase（DXS）催化MEP途径的第一步，被认为是关键的限速酶。为了证实 DXS 基因的功能 ，在本研究中，通过快速扩增 丹参叶中DNA末端，分离出 DXS的 两个同工型 （ SmDXS1 和 SmDXS2 ） 。 生物信息学分析表明，两个SmDXS与其他植物DXS具有高度同源性。组织表达模式分析表明， SmDXS1和 SmDXS2在乳 链球菌中 表现出不同的表达模式 。SmDXS1 在所有分析过的组织中表达；叶片中表达最高，其次是茎，根部表达较弱。相反， SmDXS2 转录本主要在根中检测到，但非常弱。亚细胞定位分析显示 SmDXS1 和 SmDXS2 都位于叶绿体中。 农杆菌 介导的转化用于诱导转基因毛状根系，并通过高效液相色谱法评估丹参酮的产量。与对照相比， SmDXS1和 SmDXS2的过表达显着增强了转基因根中丹参酮的积累，而仅下调了 SmDXS1 和 SmDXS2 。 SmDXS2 导致显着下降。我们的结果表明， SmDXS2 是参与丹参酮生物合成的MEP途径中的重要限速基因，并且可能是控制 丹参 毛状根培养物中丹参酮积累的有效调控靶标 。