【摘要】 为进一步推广普及烟草代谢组学研究技术,规范烟草代谢组学数据的采集流程,提高烟草代谢组学数据的可比性和可用性,建立了适用于新鲜烟叶代谢组学研究的样品采集和质谱检测方法。将采集后的鲜烟叶迅速用锡箔纸包裹并在液氮中冷冻;将充分冷冻的烟叶样品在干冰包埋下转移至实验室,真空冷冻干燥;将干燥后的烟叶样品打碎成粉末,提取代谢物;采用9种主要基于GC-MS和LC-MS技术的检测方法收集代谢组数据,分别是6种靶向检测(植物色素、游离氨基酸、有机酸、生物碱及多酚和萜类化合物)和3种非靶向检测(脂类、基于GC-MS的全成分分析和基于LC-MS全成分分析)方法。采用所建方法对不同产区、不同品种及不同生长发育时期的第十叶位鲜烟叶进行分析。结果表明:①共定性定量500余种代谢物,涉及烟草主要初生、次生代谢物,其中绝对定量101种;②主成分分析表明不同生长发育期鲜烟叶的代谢组差异较大,如大多数胺类(包括游离氨基酸)代谢物随生长发育期变化其质量分数逐渐降低;同一期鲜烟叶代谢组的生态差异大于品种差异。该方法具有稳定性和可重复性高、数据量大且可靠、覆盖代谢物多等特点,可用于烟草代谢组学研究。更多还原

【Abstract】 To promote metabolomics technology in tobacco research, to standardize tobacco metabolomics data acquisition process, and to improve data’s comparability and usability, a tobacco metabolomics workflow was established to cover fresh leave collection and mass spectrometry analysis. Fresh tobacco leaves were quickly wrapped using tin foil after picking, and then frozen in liquid nitrogen. Fully frozen tobacco leaves were embedded with dry ice and transferred to laboratories, and then grounded into powder after being lyophilized in vacuum for metabolite extraction. Tobacco metabolomics data were collected with nine different methods based mainly on GC-MS and LC-MS, including six targeted methods(on phytochromes, free amino acids, organic acids, alkaloids, polyphenols and terpenoids) and three untargeted methods(lipids, all components analysis based on GC-MS or LC-MS). Fresh tenth tobacco leaves(counted from bottom to top) sampled from different cultivars and planting areas at different growth stages were analyzed using the established methods. The results showed that: 1) Up to five hundred metabolites including mostly primary and secondary metabolites were qualified and quantified, of which 101 metabolites were absolutely quantified. 2) The metabolome of fresh tobacco leaves, indicated by principal component analysis(PCA) method, differed greatly at different growth stages, e.g. the contents of most amines(including free amino acids) in tobacco leaves gradually decreased during growing. At the same growth stage, ecological differences were greater than variety differences in the metabolome of fresh tobacco leaves. This method is stable, reliable and suitable for tobacco metabolomics research with the characteristics of high repeatability, rich data and broad coverage of metabolites in tobacco leaves.更多还原