基于LC-MS/MS分析马缨杜鹃花代谢物的变化

Analysis of metabolites change from flowering to withering of Rhododendron delavayi based on LC-MS/MS

关键词:  马缨杜鹃, 花期, 代谢组, LC-MS/MS, 代谢通路

Key words:  Rhododendron delavayi, flowering period, metabolome, LC-MS/MS, metabolic pathway

摘要:

为分析马缨杜鹃(Rhododendron delavayi)花开花至凋谢过程中的代谢产物差异及其通路,该文采用LC-MS/MS技术对其花苞期、开裂期、传粉期、盛开期、衰老期和凋谢期的化学成分进行非靶向代谢组学分析。结果表明:(1)共鉴定到973种代谢物,主要包含黄酮类、有机酸、酚酸类、氨基酸及其衍生物、脂类、生物碱等。(2)主成分分析(PCA)表明样本间代谢物存在差异,结合正交偏最小二乘判别分析(OPLS-DA)、t检验的P值和单变量分析的差异倍数(fold-change)筛选差异代谢物(VIP>1,P<0.05,Fc>2或Fc<0.5),涉及591种,在马缨杜鹃花期进入衰老期和凋谢期后差异代谢物数量和表达量显著上升,其中花苞期至开裂期差异代谢物的表达主要呈现下调,而进入衰老期和凋谢期后差异代谢物的表达主要呈现上调。(3)KEGG注释到68条代谢通路,其中差异代谢物极显著富集(P < 0.01)通路3条,包括苯丙素类生物合成、植物激素的生物合成和类黄酮生物合成。(4)结合苯丙素类、黄酮类等有效成分生物合成通路共筛选到10种代谢物包括苯丙氨酸(L-phenylalanine)、反式肉桂酸(trans-cinnamic acid)、查耳酮(chalcone)、柚皮素(naringenin)、对香豆酰基莽草酸(p-coumaroyl shikimic acid)、阿魏酸(ferulic acid)、松柏醇(coniferyl alcohol)、芥子酸(sinapic acid)、紫丁香苷(syringin)、槲皮素(quercetin)。此外,有效成分的差异代谢物表明苯丙素类生物合成代谢活动随马缨杜鹃花的发育逐渐增强,而黄酮类化合物生物合成逐渐减弱,这些关键差异代谢物可能对马缨杜鹃花的发育有重要的调控作用。该研究为马缨杜鹃花开花至凋谢进程中的有效成分代谢途径活性物质的研究提供了代谢组学基础,为进一步研究马缨杜鹃花花期调控的分子机理提供参考。

Abstract:

In order to analyze the differences of metabolites and their pathways from flowering to withering of Rhododendron delavayi, we used LC-MS/MS material separation and identification technique to non-targeted the chemical components of bud stage, dehiscence stage, pollination stage, blooming stage, senescence stage and withering stage. The results were as follows:(1)A total of 973 kinds of metabolites were detected, mainly including flavonoids, organic acids, phenolic acids, amino acids and their derivatives, lipids, alkaloids and so on.(2)Principal component analysis(PCA)showed that there were differences in metabolites among samples. Combined with orthogonal partial least squares discriminant analysis(OPLS-DA), P value of t-test and fold change of univariate analysis, differential metabolites(VIP > 1, P < 0.05, Fc > 2 or Fc < 0.5)were screened out, involving 591 species, the quantity and expression of differential metabolites increased significantly after the R. delavayi flower stage entered the senescence stage and the withering stage, in which the expression of differential metabolites from bud stage to dehiscence stage was mainly down-regulated, while those after entering senescence stage and withering stage were mainly up-regulated.(3)A total of 68 differential metabolic pathways were annotated by KEGG, of which three pathways were significantly enriched with differential metabolites(P < 0.01), including phenylpropanoids biosynthesis, plant hormone biosynthesis and flavonoid biosynthesis.(4)Based on the analysis of biosynthetic pathways of phenylpropanoids, flavonoids and other effective components, ten kinds of differential metabolites were screened from flowering to withering of R. delavayi, including L-phenylalanine, trans-cinnamic acid, chalcone, naringenin, p-coumaroyl shikimic acid, ferulic acid, coniferyl alcohol, sinapic acid, syringin and quercetin. In addition, the differential metabolites of effective components showed that phenylpropanoids biosynthesis and metabolism activities gradually increased with the development of R. delavayi, while flavonoid biosynthesis gradually decreased. These key differential metabolites may play an important role in regulating the development of R. delavayi. This study provides a metabonomic basis for the study of effective components in the metabolic pathway during the process from flowering to withering of R. delavayi, and provides a reference for the further study of the molecular mechanism of flowering period regulation of R. delavayi.