Comparative analysis of fatty acid metabolism based on transcriptome sequencing of wild and cultivated Ophiocordyceps sinensis

Abstract

Background: Ophiocordyceps sinensis is a species endemic to the alpine and high-altitude areas of the Qinghai-Tibet plateau. Although O. sinensis has been cultivated since the past few years, whether cultivated O. sinensis can completely replace wild O. sinensis remains to be determined.

Methods: To explore the differences of O. sinensis grown in varied environments, we conducted morphological and transcriptomic comparisons between wild and cultivated samples who with the same genetic background.

Results: The results of morphological anatomy showed that there were significant differences between wild and cultivated O. sinensis, which were caused by different growth environments. Then, a total of 9,360 transcripts were identified using Illumina paired-end sequencing. Differential expression analysis revealed that 73.89% differentially expressed genes (DEGs) were upregulated in O. sinensis grown under natural conditions compared with that grown under artificial conditions. Functional enrichment analysis showed that some key DEGs related to fatty acid metabolism, including acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase, were upregulated in wild O. sinensis. Furthermore, gas chromatography-mass spectrometry results confirmed that the fatty acid content of wild O. sinensis was significantly higher than that of cultivated O. sinensis and that unsaturated fatty acids accounted for a larger proportion.

Conclusion: These results provide a theoretical insight to the molecular regulation mechanism that causes differences between wild and cultivated O. sinensis and improving artificial breeding.

Keywords: Cultivated; Fatty acid metabolism; GC-MS; Ophiocordyceps sinensis; Transcriptome; Wild-grown.

Figure 1. Overview of the structure and morphology of wild-grown and cultivated O. sinensis.

(A–B, H–I), The microscopic characteristics of fruiting body and worm (middle position of structure); (C–D, F–G), The anatomical section characteristics of fruiting body and worm (middle position of structure). (E) The characteristics of appearance of O. sinensis.

Figure 2. Results of differential analysis.

(A) Volcano map of differentially expressed genes (wild vs. cultivated). (B) Heatmap of differentially expressed genes.

Figure 3. Functional annotation and enrichment results of differentially expressed genes (DEGs).

(A) Gene ontology functional enrichment map of DEGs. (B) Analysis of Kyoto Encyclopedia of Genes and Genomes significant enrichment of DEGs.

Figure 4. Metabolism and degradation of palmitic acid in O.sinesis.

(A) Degradation of palmitic acid in O.sinesis. (1) Palmitoyl coenzyme A is oxidized and decomposed to generate myristoyl-CoA. (2) Butyryl-CoA is oxidized and decomposed to produce Acetyl-CoA. (B) Heatmap of lipid-related DEGs, the green marked as a key gene in palmitic acid degradation pathway. *ACSL, long-chain acyl-CoA synthetase. CPT, carnitine O-palmitoyltransferase. ACP, acyl carrier protein.

Figure 5. QRT-PCR verification of the expressed genes in Illumina sequencing.

The expression value of β-actin was used as an inner control, the wild as a control compared to cultivated. All data are means of three replicates with error bars indicating SD.

Figure 6. The ion chromatograms of fatty acid.

(A) Total ion chromatograms of mixed fatty acid methyl ester standard. (B) The ion chromatograms of O. sinensis (OSW_S1). 1-Methyl dodecanoate (C12:0); 2-Methyl myristoleate (C14:1n5); 3-Methyl myristate (C14:0); 4-Methyl palmitate (C16:0); 5-Methyl heptadecanoate (C17:0); 6-Methyl linoleate (C18:2n6c); 7-Methyl oleate (C18:1n9c); 8-Methyl stearate (C18:0); 9-Methyl arachidate (C20:0); 10-Methyl dodecanoate (C22:0); and 11-Methyl tricosanoate (C23:0). Red numbers represent the content (mg/g) in the wild group, blue numbers represent the content in the cultivated group, “-” indicates content below the detection limit.