当归“头”“身”及“尾”部位药效取向差异的形成机制研究

Angelica sinensis Radix (ASR) is one of the most widely used herbs in traditional Chinese medicine . According to current Chinese Medicine theories, its distinct parts (head, body and tail), have different pharmacological effects resulting in their diverse clinical applications. However, the scientific explanation behind the different biological activities of different ASR parts remains unknown. One of the possible reasons could be in variation of active ingredients composition in different parts of the plant, which may be associated with the disparate expression of vital genes in biosynthetic pathways. Firstly, metabolomic and transcriptome analyses were conducted simultaneously to investigate the metabolic states of the head, body and tail parts of ASR roots. Secondly, WGCNA analyses identified the co-expression modules and hierarchical clustering constructed from metabolomic profiles in association with transcriptomic data. This was accompanied by the study of the key enzymes involved in biosynthetic pathways of main ingredients identified using chemical composition and transcriptome data for different parts. Additionally,the data of gene expression and content of ferulic acid in the above-mentioned aspects will be statistically analyzed by pearson correlation coefficient, which will be used to identify transcripts encoding enzymes involved in ferulic acid biosynthesis pathways.Finally, RACE , bacterial expression system will be employed to characterize function of ferulic acid synthetase gene. The results will provide valuable assets in aiding the comprehensive elucidation of mechanisms responsible for distinct biological activities of three parts of ASR (head, body and tail) from wholly to partly, as well as to provide basis for the rational use of different ASR parts in clinic applications. Furthermore, this project will accelerate the development of new varieties of ASR medicinal plant with improved pharmacological profiles.

当归是我国广泛使用的中药，传统中医以当归“头”、身”及“尾”分别入药，药效差异显著。该理论基于近千年中医理论的经验观察，缺乏系统的科学理论研究依据。药用植物有效成分的合成、代谢往往与关键酶基因的协同表达、调控相关。本研究以当归“头”、身”及“尾”为研究材料，构建当归不同药用部位整体差异的代谢谱及基因表达谱，通过加权基因共表达网络分析鉴定与主要差异成分高度共表达的基因模块，探讨基因模块与当归不同药用部位主要差异代谢物的互作网络；通过转录组注释预测阿魏酸合成途径酶基因簇，根据其在不同部位表达量与对应的阿魏酸含量，采用皮尔逊相关分析发掘影响当归阿魏酸差异分布的关键合成酶基因，克隆阿魏酸关键酶基因全长，利用原核表达系统对其进行功能鉴定。从整体到局部、多角度、多层次诠释当归“头”、“身”及“尾”药效取向差异的形成机制，为当归分部位入药的中医理论提供科学依据，为当归优质新品种的选育奠定一定的理论基础。

当归是我国广泛使用的中药，传统中医以当归“头”、“身”及 “尾”分别入药，药效差异显著。该理论基于近千年中医理论的经验观察，缺乏系统的科学理论研究依据。药用植物有效成分的合成、代谢与关键酶基因的协同表达、调控相关。本研究采用优化的GC-MS与UPLC-MS法分别对采自甘肃岷县的6份当归不同药用部位样品进行代谢组学分析，构建当归“头”、“身”及“尾”部位的整体代谢谱，基于以上2种检测平台与自建数据库分别检测到180、269种代谢物，鉴定其主要成分为单萜、酚酸、黄酮、木质素及香豆素及其他类。对当归不同药用部位主要代谢物含量数据进行层次聚类、正交偏二乘法判别，当归“头”、“身”及“尾”部位聚为3类，其中主要有效成分阿魏酸（指标性化合物）在“尾”中的含量显著高于“头”与“身”。采用代谢组及转录组联合分析当归不同药用部位化合物的代谢差异，通过加权基因共表达网络分析鉴定了与主要差异成分高度共表达的基因模块，注释并预测阿魏酸合成途径酶基因簇，根据阿魏酸含量及生物合成相关基因表达量数据，采用皮尔逊相关分析发掘影响当归阿魏酸差异分布的关键合成酶基因，表明PAL、C3H、CQT是合成阿魏酸的关键酶基因。荧光Real-time PCR结果显示以上3种基因在当归“尾”中表达量最高，“头”、“身”中次之，该结果与阿魏酸成分在当归不同药用部位中的HPLC含量测定结果一致，预测PAL、C3H、CQT蛋白在当归有效成分阿魏酸的合成过程中可能发挥了作用。进一步筛选并克隆获得当归ASC3H全长基因，开放阅读框长度为1530bp，编码509个氨基酸，含细胞色素P450的保守结构域，多重氨基酸序列比对分析结果显示ASC3H与其他植物的C3H基因具有较高相似性。利用原核表达系统对C3H蛋白进行酶促反应从而鉴定其基因功能，结果表明C3H蛋白可能在阿魏酸合成中间产物途径中起关键限速酶作用。本研究从整体到局部、多角度、多层次诠释当归“头”、“身”及“尾”药效取向差异的形成机制，为今后通过基因工程手段合成阿魏酸目标产物或培育优质当归品种奠定了研究。