IPP转运影响雷公藤甲素累积的分子机制研究

Triptolide is an abietane diterpenoid with significant anti-inflammatory and anti-tumor bioactivity, but its content is very low in Tripterygium wilfordii Hook. f.. The biosynthesis mechanism of triptolide is not clear at present. The precursor of triptolide is IPP which provided by MVA (Site: cytoplasm) and MEP (Site: plastid) pathways and widely accepted as the common intermediate between the two pathways. It also can freely exchange between the two pathways by penetrating lipid bilayer. Its source and transport processes affect the accumulation of terpenoids. Our previous work showed that if the MEP pathway was only inhibited, the accumulation of triptolide was not decreased. In this condition, we hypothesized that MVA and MEP pathways may participate in the biosynthesis and accumulation of triptolide through IPP translocation..This project is intended to take the IPP translocation as a key point. Metabolic pathway regulation and 13C stable isotope labeling will be used as experimental methods, and the qualitative and quantitative analysis will be completed by UPLC-MS and C-NMR. In the end, the synthesis time, translocation time, translocation pathway and translocation rate of IPP during the accumulation of triptolide will be determined, the contribution of MVA and MEP pathway to the accumulation of triptolide will be analyzed, and the internal relationship among IPP translocation, key enzyme genes in MVA/MEP pathway, and triptolide accumulation will be explored. It is the first time to elucidate the molecular mechanism of IPP translocation affecting on triptolide accumulation. These results will provide scientific basis in improving the theory of triptolide biosynthesis, and provide new ideas and methods for regulating triptolide accumulation.

雷公藤甲素是一种具有显著抗炎、抗肿瘤活性，但在植物中含量极低的二萜类化合物，目前生物合成机制尚不明确。异戊烯基焦磷酸（IPP）为萜类化合物共同前体，经由MVA和MEP途径生成，并能透过磷脂双分子层在两途径间自由转运，其来源和转运过程影响萜类化合物的累积。前期研究表明单一抑制MEP途径并不能导致雷公藤甲素累积量的下降，预示着MVA和MEP途径可能通过IPP转运而共同参与雷公藤甲素的累积。本项目拟以IPP转运过程为切入点，通过代谢途径调控和13C稳定同位素标记法，经UPLC-MS和C-NMR的定性、定量分析，明确雷公藤甲素累积过程中IPP合成时间、IPP转运时间、路径和转运率，解析MVA和MEP途径对雷公藤甲素累积的贡献能力，探究IPP转运、MVA和MEP途径关键酶基因和雷公藤甲素累积三者间的内在联系，进而首次阐明IPP转运影响雷公藤甲素累积的分子机制，为调控雷公藤甲素累积提供新的思路和方法。

雷公藤甲素是一种具有显著抗炎、抗肿瘤活性，但在植物中含量极低的二萜类化合物，目前生物合成机制尚不明确。异戊烯基焦磷酸（IPP）为萜类化合物共同前体，经由MVA和MEP途径生成，并能透过磷脂双分子层在两途径间自由转运，其来源和转运过程影响萜类化合物的累积，但关于雷公藤甲素累积过程中IPP来源和转运的研究尚未开展。因此，本项目以揭示雷公藤甲素累积过程中IPP形成、转运的特点和机制为研究目标，运用代谢途径调控、关键功能基因挖掘等方法对其进行研究，阐明了雷公藤甲素累积过程中，IPP转运的特点及相关机制，并成功筛选得到了5条影响IPP形成的关键MYB候选转录因子。取得的研究成果如下：1. 解析了不同诱导子对雷公藤甲素生物合成途径的调控作用，如：美伐他汀（MVA途径抑制剂）、磷铵霉素钠（MEP途径抑制剂）、DLG（IPP转运抑制剂）和独脚金内酯类似物（GR24）可不同程度地抑制雷公藤甲素的累积；2. 明确了IPP转运在雷公藤甲素生物合成过程中的时空特点及相关分子机制，即在雷公藤悬浮细胞培养早期，IPP转运主要经由质体（MEP途径）转运到细胞质（MVA途径），而细胞培养晚期则相反，且IPP转运可影响雷公藤甲素上游关键酶基因（TwDXR、TwHDR、TwHMGR、TwHMGS）的表达；3. 完成了雷公藤MYB转录因子家族生物信息学分析，并筛选得到了6条对IPP形成起关键作用的候选转录因子（TwMYB33、TwMYB34、TwMYB45、TwMYB67、TwMYB102和TwMYB103）。相关研究成果既填补了雷公藤甲素生物合成过程中IPP转运机制方面的空白，又多角度、全面地阐释雷公藤甲素生物合成过程中的关键要素，对于理清雷公藤甲素生物合成途径，提升雷公藤中药资源的研究水平具有重要意义。在本项目的资助下，共发表学术论文2篇，申请国家发明专利1项，项目资助期间，项目负责人荣获2019年中国药学会科学技术奖三等奖（排名5/6），协助指导在读硕士研究生1名。