10-HGO催化的环化反应高效调控紫草宁合成的生化及分子机制解析

Shikonin and its derivatives have significant medicinal and industrial values. However, it remains unknown about the regulatory enzyme that catalyzes the key cyclization reaction step in the biosynthesis pathway of shikonin and its derivatives formation. In previous study, we discovered a candidate key enzyme (10-hydroxygeraniol oxidoreductase) gene 10-HGO possibly catalyzing this step. The expression of this gene is significantly up-regulated in the cells with shikonin formation, while there is no expression in the cells without shikonin formation. To deeply dissect the biochemical and molecular characteristics of this gene, and the functional mechanism of the cyclization reaction catalyzed by this gene in efficiently regulating shikonins formation, the cells/hairy roots of boraginaceae plants, such as Arnebia euchroma and Lithospermum erythrorhizon, will be used as materials to clone the full-length cDNA of this gene by using RACE method; clarify the relation between shikonin formation and enzyme activity, protein and gene expression patterns; in vitro eukaryotically express and purify the enzyme protein, and clarify the biochemical characteristics of the substrate cyclization reaction catalyzed by it; heterologous complementation analysis; functional analysis of core site mutations; protein-protein interaction analysis; transgenic analyses (RNAi and OE) and metabolomics analysis (LC-MS, NMR, etc. ) of the substrates and products, to collaboratively dissect the function of 10-HGO gene. The results of this project will not only lay foundation for further revealing the biochemical and molecular regulatory mechanism of shikonin formation, but also provide key target for bioengineering practice of natural product biosynthesis of boraginaceae plants and other non-model plants.

紫草宁及其衍生物具有重要的药用和工业价值，然而控制其生物合成途径中关键环化反应这一步骤的催化酶却一无所知。在前期的研究中我们发掘了一个可能催化该步骤的关键酶（10-羟基香叶醇氧化还原酶）基因10-HGO，该基因在细胞合成紫草宁时超强表达，而不合成紫草宁时则无任何表达。为深入解析该基因的生化和分子特征及其催化的环化反应高效调控紫草宁合成的作用机制，本项目拟以紫草细胞/毛状根为材料，RACE法克隆其cDNA全长；明确其拷贝数等分子特征；明确酶活性、蛋白和基因表达模式与紫草宁合成的相关性；体外真核表达、纯化酶蛋白并阐明其催化底物环化反应的生化特征；异源表达及功能互补分析等；转基因（RNAi和OE）及代谢组学（LC-MS、NMR等）的底物和产物分析等协同解析其功能。本项目的完成可为深入揭示紫草宁合成调控的生化和分子机制奠定理论基础，并为紫草等非模式植物的次生代谢生物工程实践提供关键靶标。

紫草作为一种重要的传统药用植物，其根系或组织培养体系（如愈伤组织细胞、茎段和毛状根）可合成具有重要药用价值的次生代谢产物-紫草宁及其衍生物。尽管紫草宁的主体合成途径已基本清楚，但一些关键酶促途径仍知之甚少。本项目以紫草细胞和/或毛状根为材料，发掘了一些重要的酶基因，并通过生化和转基因策略等解析了其调控效应，获得了相关的转基因毛状根株系。本项目研究结果为深入揭示紫草宁合成调控的分子机制，以及非模式植物的次生代谢生物工程实践奠定了基础。主要结果概述如下：.（1）成功克隆了紫草中的酶基因10-HGO的cDNA全长；（2）通过转录组和real-time PCR分析了10-HGO的表达调控模式；（3）构建了10-HGO的异源昆虫表达体系，成功的获得了10-HGO蛋白；（4）体外生化功能分析表明，10-HGO可成功地将10-hydroxygeraniol催化成10-oxogeranial，表明其主要功能之一可能是负责环化反应前的羟基羰基化过程；（5）通过转基因策略成功地获得了10-HGO的过表达（Overexpression）和RNA干扰（RNAi）毛状根株系；（6）进一步通过拟南芥异源表达体系的转录组和代谢组学分析，探讨了10-HGO对次生代谢的调控效应；（7）结合文献深入分析了转录组数据，挖掘了两个可能在环化反应中起关键作用的候选基因IDS和BBE，正在深入研究，有待后续补充研究成果；（8）发表了标注资助号的SCI论文10篇，中文期刊论文1篇，会议论文5篇；申请发明专利1项。