Integrated transcriptomic and metabolomic analysis reveals stage-associated molecular profiles in Ophiocordyceps sinensis

Abstract

Ophiocordyceps sinensis is globally recognized for its exceptional nutritional and medicinal properties. Variations in the edible qualities and tonic values of O. sinensis at different harvesting stages remain poorly understood in terms of compositional changes and regulatory mechanisms. Utilizing UPLC-MS/MS and transcriptome sequencing (RNA-seq), this study unveiled discrepancies in metabolite accumulation and gene expression of O. sinensis across various harvesting stages. Metabolomics analysis identified 596 differentially accumulated metabolites (DAMs), primarily enriched in amino acid-related metabolic pathways such as tyrosine, tryptophan, cysteine, and methionine metabolism. The up accumulation of organic acids and derivatives with delayed harvesting led to distinct abundances and compositions of amino acids, peptides, analogs, and fatty acids and conjugates, ultimately influencing the quality of O. sinensis. Transcriptomic analysis revealed 2550 differentially expressed genes (DEGs) at different harvesting stages, with KEGG-based enrichment analysis highlighting their involvement in amino acid-related activities like tyrosine metabolism and fatty acid degradation. The upregulation of these DEGs in amino acid-related pathways presents a promising target for studying O. sinensis quality. Integrative metabolomic and transcriptomic analyses indicated potential roles for DDC (G6O67_000335), TYR (G6O67_005660), AOC (G6O67_005457), and fahA (G6O67_004634) in the synthesis pathways of amino acids, peptides, and their analogs, suggesting a possible indirect association with O. sinensis quality. These findings offer novel insights into the molecular mechanisms underlying the quality formation and metabolic evolution of O. sinensis.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12864-025-11869-3.

Keywords: Ophiocordyceps sinensis; Harvesting stages; Metabonomics; Nutritional ingredient; Transcriptomics.

Fig. 1

Ophiocordyceps sinensis harvested at different stages. D1: sclerotia germination stage, D2: sclerotia development stage, D3: mature stage of sclerotia

Fig. 2

Metabolomic analysis of O. sinensis at different harvest stages. (A) PCA score plot. (B) O. sinensis upregulated and downregulated the number of DAMs at the different harvest stages. (C) Upset diagram of O. sinensis at different harvest stages. The X axis represents the number of DAMs, the Y axis represents different comparison groups, and the points of different colors represent the common features of different comparison groups. (D: D1 vs D2, E: D2 vs D3, and F: D1 vs D3). KEGG pathway enrichment maps of DAMs in O. sinensis at different harvest stages. The DA Score reflects overall changes in the DAMs pathway. DA Score > 0 indicated that the expression trend of DAMs in the pathway was upregulated. DA Score < 0 indicates that the expression of DAMs in the pathway is downregulated. The length of the line segment indicated the absolute value of the DA Score. The blue font represents the common enrichment pathway of the three comparison groups. ‘*’ represents a significant difference at the p < 0.05 level, ‘**’ represents a significant difference at the P < 0.01 level

Fig. 3

Cluster analysis of DAMs. A K-means clustering analysis of the DAMs, where the total represents the number of DAMs in this subclass. B Hierarchical cluster analysis of the top 100 VIP DAMs, the name of which is represented by metabolite ID

Fig. 4

Transcriptome analysis of O. sinensis at different harvest stages. A PCA score map of gene expression in three groups of samples. B Correlation diagram of three biological replicates of three groups of samples. C The number of DEGs in the three comparison groups. D The number of annotations of all expressed genes in different databases. E The upset map of the number of DEGs in the three comparison groups. The X axis represents the number of DAMs, the Y axis represents different comparison groups, and the points of different colors represent the common features of different comparison groups. F K-means cluster analysis of all DEGs. G GO annotation of repeated DEGs in the three comparison groups. H KEGG enrichment analysis of overlapping DEGs in the three comparison groups

Fig. 5

Based on qRT-PCR, the expression analysis of 29 DEGs in 3 harvest stages was determined. Different uppercase letters represent significant differences in qRT-PCR values at the p < 0.05 level, and different lowercase letters represent significant differences in RNAseq values at the p < 0.05 level. The error bar represents the standard deviation of three biological replicates

Fig. 6

Integrative analysis of DAMs and DEGs. (A, D1 vs. D2; B, D2 vs. D3; C, D1vs D3) Enrichment map of KEGG pathway co-mapped by DAMs and DEGs. (D) DAMs and DEGs map KEGG pathway enrichment maps shared by all comparison groups

Fig. 7

Tyrosine metabolism analysis. A Correlation network diagram of DAMs and DEGs involved in tyrosine metabolism. B Comparison of DAMs and DEGs in tyrosine metabolism based on KEGG database. Red fonts represent significantly enriched DAMs, and blue fonts represent important DAMs. The virtual line connected to the metabolic pathway represents the input or output of metabolites from this metabolic pathway. The blue italic character on the path edge represents the down-regulated gene, the red character represents the up-regulated gene, and the black character represents the gene with no significant difference