滇龙胆中龙胆苦苷生物合成与积累特征的研究

Gentiana rigescens Franch, considered to have medicinal uses in Yunnan province, is a priority for development. However, the content of gentiopicroside in cultivated material prepared for medicines is low, with inconsistent quality. Building on our previous research, this project aims to further understanding of the gentiopicroside biosynthesis pathway. The proposed research will focus on the following two factors influencing the biosynthesis and accumulation of gentiopicroside in plants: (1) the effect of the MEP pathway and the MVA pathway, which are involved upstream in gentiopicroside biosynthesis; (2) the dynamics and distribution of the end products of MEP and MVA pathways (secoiridoid compounds such as gentiopicroside) among plant tissues (leaf and root) in different seasons. Research methods from different fields will be used in this project, such as transcriptomics and plant anatomy metabolomics. In the project, we propose to study the accumulation sites of the active compounds. Further, the process of biosynthesis and accumulation of gentiopicroside will be studied from the two aspects of rooting and sprouting processes. Meanwhile, the relative effects of the MEP and MVA pathways will be analyzed by positive regulation of the upstream pathway and feedback regulation of the downstream pathway. Further, the dynamic changes of active compounds and the mechanism of upstream pathway regulation of metabolite synthesis in plant tissues in different growth stages will be clarified, from the perspective of temporal dynamics and static plant organs. The results of this project will provide a reference foundation for approaches to enhancing the accumulation of active compounds and quality control of G. rigescens.

滇龙胆（Gentiana rigescens Franch）为云南省重点发展的药用作物，是云南道地药材。然而，栽培药材龙胆苦苷含量低，品质不均一。本项目拟在前期研究基础上，以探明龙胆苦苷生物合成途径为目标，针对影响其合成与积累的两个方面①龙胆苦苷的上游合成途径MEP途径和MVA途径的作用；②MEP和MVA途径的末端产物（龙胆苦苷等裂环烯醚萜类化合物）在不同器官（枝叶和根）、不同季节中的动态分配。利用植物解剖学、代谢组学、转录组学等多学科交叉方法，通过有效成分组织定位，从生根和发芽两方面研究龙胆苦苷合成积累规律，同时通过上游正向调控和下游反馈调节两方式分析MEP和MVA途径的作用比例，进一步探讨有效成分的时空变化，从动态和静态两种角度探明上游合成途径对代谢的调控机制，为提高滇龙胆有效成分、精准质量控制等生产调控提供理论基础和参考。

滇龙胆（Gentiana rigescens Franch）是云南省重点发展的药用作物，是云南省道地药材。然而栽培滇龙胆存在龙胆苦苷含量低，品质不均一等问题，因此研究滇龙胆中龙胆苦苷等有效物质积累，解析其生物合成途径，对滇龙胆品质提升具有重要意义。本研究表明龙胆苦苷主要积累在根的薄壁组织中，花期及花期前后直接参与龙胆苦苷生物合成的关键酶基因在叶片中表达量高于根部，根内与萜类及龙胆苦苷生物合成相关的差异代谢物和差异基因在“开花前VS开花期”比“开花后VS开花期”更为集中，有效成分獐牙菜苷在花期和花后期的含量呈现减少趋势，开花前VS开花期神经酰胺酶（CERS）与獐牙菜苷呈正相关，推测CERS可能与龙胆苦苷生物合成途径有关。热刺激后龙胆苦苷生物合成途径中关键酶基因甲羟戊酸二磷酸脱羧酶、4-羟基-3-甲基-2-乙烯基焦磷酸还原酶、细胞色素P450 13-1的表达量上调，可能是热刺激下促使龙胆苦苷含量提高的关键基因。发现滇龙胆苗内龙胆苦苷的积累及分解周期约为3天，利用外源激素水杨酸、抑制剂洛伐他汀和膦胺霉素处理滇龙胆苗，并对关键酶基因相对表达量及龙胆苦苷含量进行分析研究后发现，2-C-甲基-D-赤藓糖醇-4-磷酸酯（MEP）途径是龙胆苦苷生物合成的主要来源，但甲羟戊酸（MVA）和MEP途径之间可能存在串扰。在此基础上发现绿光照射可有效提高滇龙胆中龙胆苦苷的积累，热烫最优工艺为热烫时间5 min、热烫温度40℃、烘干温度60℃。本项目围绕龙胆苦苷生物合成途径开展相关研究，旨在为提高滇龙胆药材中龙胆苦苷含量，建立精细化、精准化生产提供理论依据和参考。已发表文章3篇，录用1篇，申请发明专利3项（授权1项）、培养博士1名，助理研究员1名、副研究员1名，部分研究成果得到应用推广，实现销售额1200万元。