氨基酸靶向代谢组学分析晶粒小鬼伞自溶过程关键差异代谢物

Analysis of key differential metabolites in the autolysis process of Coprinellus micaceus by amino acid targeted metabolomics

中文关键词:  晶粒小鬼伞  自溶  氨基酸代谢  靶向代谢组学

英文关键词:Coprinellus micaceus  autolysis  amino acid metabolism  targeted metabolomics

中文摘要:

      目的 通过氨基酸靶向代谢组学, 筛选晶粒小鬼伞自溶过程中的氨基酸差异代谢物, 并分析其自溶过程潜在通路。方法 采集晶粒小鬼伞样本, 将样本分成对照组和自溶组, 每组样本各6份, 共12份。采用超高效液相色谱-串联质谱方法检测样本中的24种氨基酸, 通过多元统计主成分分析(principal component analysis, PCA)评估样本重复性与整体代谢差异趋势, 正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA)最大化组间差异, 筛选差异代谢物, 并对差异代谢物进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路富集分析。结果 通过PCA和OPLS-DA分析, 主成分1 (PC1)的贡献率为52.6%, 主成分2 (PC2)的贡献率为24.1%, 自溶组(R)和对照组(W)间完全分离, 晶粒小鬼伞在自溶过程中的氨基酸代谢物表现出显著差异; 以变量重要性投影(variable importance projection, VIP )≥1, P<0.05且|log2FC|≥0.58为条件筛选差异代谢物, 晶粒小鬼伞自溶过程中的差异氨基酸为鸟氨酸下调2.7倍, 色氨酸、瓜氨酸和半胱氨酸分别上调1.8倍、2.5倍和1.9倍; 聚类分析结果显示, 自溶组和对照组的氨基酸代谢物存在显著差异, 半胱氨酸、色氨酸和瓜氨酸在自溶组中呈显著高表达, 鸟氨酸呈显著低表达; Pearson相关性分析显示, 半胱氨酸、瓜氨酸与色氨酸之间呈极显著正相关(P<0.01), 而三者与鸟氨酸均呈显著负相关(P<0.05)。对存在显著差异的氨基酸代谢物进行富集分析, 结果显示, 晶粒小鬼伞自溶的潜在核心高富集通路为氨基酸生物合成通路, 次核心高富集通路为植物次生代谢物的生物合成通路, 中等富集通路为精氨酸生物合成、甘氨酸丝氨酸和苏氨酸代谢、谷胱甘肽代谢、2-氧羧酸代谢、癌症中的中心碳代谢、氨酰基转移核糖核酸生物合成、D-氨基酸代谢、蛋白质消化与吸收和辅因子生物合成。结论 筛选确定鸟氨酸、色氨酸、瓜氨酸和半胱氨酸为晶粒小鬼伞自溶过程中的关键差异代谢物; 上述4种差异代谢物可通过多条潜在通路协同调控晶粒小鬼伞自溶进程。本研究结果可为大型真菌保鲜贮藏、加工工艺优化及资源综合开发利用提供数据支撑与科学依据。

英文摘要:

      Objective To screen the differential metabolites of amino acids during the autolysis process of the Coprinellus micaceus by amino acid targeted metabolomics, and analyze the potential pathways of its autolysis process. Methods The samples of Coprinellus micaceus were collected, the samples were divided into a control group and an autolysis group, with 6 samples in each group, totaling 12 samples. The 24 kinds of amino acids in the samples were detected by ultra-performance liquid chromatography-tandem mass spectrometry. The sample repeatability and the trend of overall metabolic differences were evaluated through multivariate statistical principal component analysis (PCA). Orthogonal partial least squares discriminant analysis (OPLS-DA) maximizes differences between groups and screens for differential metabolites and the Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was conducted for the differential metabolites. Results Through PCA and OPLS-DA, the contribution rate of principal component 1 (PC1) was 52.6%, and that of principal component 2 (PC2) was 24.1%. The autolysis group (R) and the control group (W) were completely separated, and the amino acid metabolites of Coprinellus micaceus disseminatus during autolysis showed significant differences. Differential metabolites were screened under the conditions of variable importance projection (VIP)≥1, P<0.05, and |log2FC|≥0.58. The differential amino acids during the autolysis of Coprinellus micaceus were ornithine downregulated by 2.7-fold, and tryptophan, citrulline and cysteine upregulated by 1.8-fold, 2.5-fold and 1.9-fold, respectively. Cluster analysis results showed significant differences in amino acid metabolites between the autolysis group and the control group. Cysteine, tryptophan, and citrulline were significantly highly expressed in the autolysis group and stably lowly expressed in the control group, while the expression pattern of ornithine was completely opposite to that of the 3 metabolites. Pearson correlation analysis showed that there were extremely significant positive correlations between cysteine, citrulline and tryptophan (P<0.01), while all 3 showed significant negative correlations with ornithine (P<0.05). Enrichment analysis was performed on the significantly different amino acid metabolites. The results showed that the potential core highly enriched pathway in the autolysis of Coprinellus micaceus was the amino acid biosynthesis pathway, the sub-core highly enriched pathway was the biosynthesis of plant secondary metabolites and the moderately enriched pathways included arginine biosynthesis, glycine, serine and threonine metabolism, glutathione metabolism, 2-oxocarboxylic acid metabolism, central carbon metabolism in cancer, aminoacyl-tRNA biosynthesis, D-amino acid metabolism, protein digestion and absorption, and cofactor biosynthesis. Conclusion Ornithine, tryptophan, citrulline and cysteine are screen and identify as the key differential metabolites in the autolysis process of Coprinellus micaceus. These 4 kinds of amino acid differential metabolites can synergistically regulate the autolysis process of Coprinellus micaceus through multiple potential pathways. The results of this study can provide data support and scientific basis for the preservation and storage of macrofungi, optimization of processing techniques, and comprehensive development and utilization of resources.