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. 2018 Dec 19;5(12):181247.
doi: 10.1098/rsos.181247. eCollection 2018 Dec.

Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae

Tengfei Liu  1 , Ziyao Liu  1 , Xueyan Yao  2 , Ying Huang  1 , Qingsong Qu  1 , Xiaosa Shi  1 , Hongmei Zhang  1 , Xinyuan Shi  1
Affiliations

Identification of cordycepin biosynthesis-related genes through de novo transcriptome assembly and analysis in Cordyceps cicadae

Tengfei Liu et al. R Soc Open Sci. .

Abstract

Cordyceps cicadae (Chanhua) is a parasitic fungus that grows on Cicada flammata larvae and is used to relieve exhaustion and treat numerous diseases, in part through its active constituent, cordycepin. We used de novo Illumina HiSeq 4000 sequencing to obtain transcriptomes of C. cicadae mycelium, fruiting body, and sclerotium, and identify differentially expressed genes. In the mycelium versus sclerotium libraries, 1576 upregulated and 2300 downregulated genes were identified. In the mycelium versus fruiting body and fruiting body versus sclerotium body libraries, 1604 and 1474 upregulated and 1365 and 1320 downregulated genes, respectively, were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified 19 genes differentially expressed in mycelium versus fruiting body as related to the purine pathway, along with 28 and 16 genes differentially expressed in the mycelium versus sclerotium and fruiting body versus sclerotium groups, respectively. Gene expression of six key enzymes was validated by quantitative polymerase chain reaction. Specifically, 5'-nucleotidase (c62060g1) and adenosine deaminase (c35629g1) in purine nucleotide metabolism, which are involved in cordycepin biosynthesis, were significantly upregulated in the sclerotium group. These findings improved our understanding of genes involved in the biosynthesis of cordycepin and other characteristic secondary metabolites in C. cicadae.

Keywords: Cordyceps cicadae Shing; RNA-Seq; artificial culture; cordycepin; de novo transcriptome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
The analogues of cordycepin and the putative biosynthetic pathways of cordycepin: (a) chemical structure of cordycepin analogues. (b) AMPD, AMP deaminase; ADEK, adenylate kinase; ADK, adenosine kinase; RNR, ribonucleotide reductase; PNP, purine nucleoside phosphorylase; NT5E, 5′-nucleotidase;Cns1, oxidoreductase/dehydrogenase; Cns2, metal-dependent phosphohydrolase; Cns3/NK, N-terminal: nucleoside/nucleotide kinase (NK); C-terminal: HisG domain for ATP. The straight lines show that synthetic pathways have been validated; the dashed lines show that synthetic pathways were predicted.
Figure 2.
Figure 2.
The fruiting body development of C. cicadae Shing: (a) Chinese Tussah silkmoth pupae were inoculated with conidia from the C. cicadae Shing strain and incubated for 7 days in the dark. (b–d) Infected Chinese Tussah silkmoth pupae were cultured with a 16 L : 8 D cycle at 500 lx and relative humidity greater than 80% to form sclerotium and fruiting bodies (b, 3 days; c, 6 days; d, 21 days). (e) The infected pupae were embedded in glass bottles with sterile wet soil and cultured with a 16 L : 8 D cycle at 500 lx and relative humidity greater than 80% for 21 days. (f) The artificially cultured C. cicadae were divided into the sclerotium and fruiting body.
Figure 3.
Figure 3.
Distribution of the homology search against the NCBI database: (a) Venn diagram of the number of orthologous unigenes. (b) Distribution of E-value. (c) Similarity of expressed sequence tags against the NR database. (d) Distribution of annotated species.
Figure 4.
Figure 4.
GO and KEGG analysis. GO classification of the C. cicadae unigene library. Histogram showing the classification of eukaryotic orthologous groups (KEGG) of the annotated unigenes.
Figure 5.
Figure 5.
Gene expression profiles in the mycelium, fruiting body and sclerotium of artificially cultured Cordyceps cicadae Shing. (a) The numbers of up- and down-regulated genes are shown in pairwise comparisons of the three libraries. (b) Venn diagram of the number of DEGs based on three comparisons of mycelium versus fruiting body (MF), fruiting body versus sclerotium (FS) and mycelium versus sclerotium (MS).
Figure 6.
Figure 6.
Expression profiles of six candidate genes as revealed by qPCR (left y-axis) and RNA-Seq (right y-axis) during the three developmental stages: the mean ± s.e. represents three biological replicates. Unigene IDs are marked above each figure. (a) c19459g1 (ribonucleoside-diphosphate reductase subunit M1). (b) c34980g1 (adenosine deaminase CECR1). (c) c19447g1 (5'-nucleotidase). (d) c35629g1 (adenosine deaminase ADA2). (e) c62060g1 (5'-nucleotidase). (f) c76121g1 (nucleoside-diphosphate kinase). Different numbers of asterisks represent significant differences between groups (*p < 0.05 ,**p < 0.01 ,***p < 0.0001).
Figure 7.
Figure 7.
Putative biosynthesis pathway for cordycepin in C. cicadae.
See this image and copyright information in PMC

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