天然产物研究与开发 ›› 2022, Vol. 34 ›› Issue (4): 553-562.doi: 10.16333/j.1001-6880.2022.4.002

• 研究论文 • 上一篇    下一篇

基于广泛靶向代谢组学技术与高通量测序技术探究广陈皮陈化机制

胡   媛1,吴   蓓1,易   达2,陈鸿平1,杨放晴1,王   福1*,刘友平1*   

  1. 1成都中医药大学药学院 西南特色中药资源国家重点实验室,成都 611137;2 南充市食品药品检验所,南充 637000
  • 出版日期:2022-04-29 发布日期:2022-04-29
  • 基金资助:
    国家自然科学基金(81973436,82104340)

Integration of high throughput sequencing and widely targeted metabolomics reveals the aging mechanism of Pericarpium Citri Reticulatae 'Chachiensis'

HU Yuan1,WU Bei2,YI Da2,CHEN Hong-ping1, YANG Fang-qing1,WANG Fu1*,LIU You-ping1*   

  1. 1State Key Laboratory of Southwestern Chinese Medicine Resources,Department of Pharmacy,Chengdu University of TCM,Chengdu 611137,China;2 Nanchong Institute for Food and Drug Control,Nanchong 637000,China

  • Online:2022-04-29 Published:2022-04-29

摘要:

采用广泛靶向代谢组学技术与高通量测序技术探究广陈皮共存微生物对次生代谢产物的影响规律,为广陈皮的陈化机制提供科学依据。运用Waters ACQUITY UPLC HSS T3 C18色谱柱(1.8 μm,2.1 mm×100 mm),流动相0.04%乙酸水溶液-乙腈梯度洗脱,流速0.4 mL/min,选择电喷雾离子源在正、负离子模式下进行采集检测;采用CTAB法对口腔拭子样品进行DNA提取,建库试剂盒进行文库构建,构建好的文库经Qubit和Q-PCR定量,使用HiSeq 2500 PE250进行上机测序;对代谢组检测出的差异代谢物和高通量测序比对出的差异微生物进行相关性分析。结果表明共鉴定出386种次生代谢产物,包括酚酸类79种,黄酮类143种,蒽醌类4种,木脂素与香豆素17种,鞣质3种,生物碱47种,萜类6种,其他类87种。主成分分析(PCA)与热图分析(Heatmap)结果表明贮藏1年与贮藏3年样品次生代谢物含量存在明显差异,可明显区分,共筛选出79种差异代谢物;高通量测序基因序列与微生物参考数据库进行比对可得到共58个属,贮藏1年与贮藏3年样品微生物菌落结构相似,但相对丰度与优势菌属不同。差异代谢物与差异微生物关联分析表明,青霉属、曲霉属、耐干霉菌等优势真菌可显著影响广陈皮中黄酮类成分以及有香味化合物含量。广陈皮在陈化过程中优势微生物菌落结构的改变导致药材活性成分以及香气成分含量的变化可能是其陈化机制之一。

关键词: 广陈皮, 代谢组, 高通量测序, 相关性分析, 陈化机制

Abstract:

Widely targeted metabonomics and high-throughput sequencing technologies were used to investigate the effects of coexisting microorganisms on secondary metabolites in Pericarpium Citri Reticulatae ‘Chachiensis’ (PCRC),which will provide a scientific basis for the aging mechanism of PCRC.A Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm,2.1 mm×100 mm) was used with 0.04% acetic acid-acetonitrile gradient elution as mobile phase at a flow rate of 0.4 mL/min.Electrospray ionization was selected for collection and detection in positive and negative ion modes.DNA was extracted from oral swab samples by CTAB method,and library construction kit was used for library construction.The library was quantified by Qubit and Q-PCR,and sequenced by HiSeq 2500 PE250.Correlation analysis was conducted between the differential metabolites and the differential microorganisms.The results showed that 386 kinds of secondary metabolites were identified,including 79 phenolic acids,143 flavonoids,4 anthraquinones,17 lignans and coumarins,3 tannins,47 alkaloids,6 terpenoids and 87 other kinds.Principal component analysis and heatmap analysis showed that the contents of secondary metabolites in the samples stored for 1 year and 3 years were significantly different.A total of 58 genera were obtained by comparing the gene sequences of high-throughput sequencing with the microbial reference database.The bacterial colonies of the samples stored for 1 year and 3 years were similar in structure,but the relative abundance was different from that of the dominant genus.The correlation analysis of differential metabolites and differential microorganisms showed that the dominant fungi,such as Penicillium and Aspergillus could significantly affect the content of flavonoids and aromatic compounds.The aging mechanisms may be closely related to the change of active components and aroma components affected by the coexisting microorganisms.

Key words: Pericarpium Citri Reticulatae 'Chachiensis', metabolomics, high-throughput sequencing, correlation analysis, aging mechanism 

中图分类号:  R28