High Throughput Identification of the Potential Antioxidant Peptides in Ophiocordyceps sinensis.

Ophiocordyceps sinensis, an ascomycete caterpillar fungus, has been used as a Traditional Chinese Medicine owing to its bioactive properties. However, until now the bio-active peptides have not been identified in this fungus. Here, the raw RNA sequences of three crucial growth stages of the artificially cultivated O. sinensis and the wild-grown mature fruit-body were aligned to the genome of O. sinensis. Both homology-based prediction and de novo-based prediction methods were used to identify 8541 putative antioxidant peptides (pAOPs). The expression profiles of the cultivated mature fruiting body were similar to those found in the wild specimens. The differential expression of 1008 pAOPs matched genes had the highest difference between ST and MF, suggesting that the pAOPs were primarily induced and play important roles in the process of the fruit-body maturation. Gene ontology analysis showed that most of pAOPs matched genes were enriched in terms of 'cell redox homeostasis', 'response to oxidative stresses', 'catalase activity', and ' integral component of cell membrane'. A total of 1655 pAOPs was identified in our protein-seqs, and some crucial pAOPs were selected, including catalase, peroxiredoxin, and SOD [Cu-Zn]. Our findings offer the first identification of the active peptide ingredients in O. sinensis, facilitating the discovery of anti-infectious bio-activity and the understanding of the roles of AOPs in fungal pathogenicity and the high-altitude adaptation in this medicinal fungus.

[Comparison of endogenetic microbial community diversity between wild Cordyceps sinensis,artificial C. sinensis and habitat soil].

To obtain the difference of the fungal and bacterial community diversity between wild Cordyceps sinensis, artificial C. sinensis and their habitat soil, Illmina Hiseq high-throughput sequencing technology was applied. The results show that Proteobacteria was the dominant bacterial phylum in C. sinensis, Actinobacteria was the dominant bacterial phylum in soil microhabitat, Ophiocordyceps sinensis was the predominant dominant fungus of C. sinensis. The α diversity analysis showed that the fungal diversity of stroma was lower than other parts, and the fungal diversity of wild C. sinensis was lower than that of artificial C. sinensis. The ß diversity analysis showed that the fungal and bacterial community diversity of soil microhabitat samples was significantly different from that of C. sinensis. The fungal community diversity was less different between wild and artificial C. sinensis, especially in sclerotia. LEfSe analysis showed a lot of species diversity between wild and artificial C. sinensis. Those different species between wild C. sinensis, artificial C. sinensis and their habitat soil provide ideas for further research on breed and components of C. sinensis.

mRNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms regulating induction of fruiting body in Ophiocordyceps sinensis.

Ophiocordyceps sinensis has been a source of valuable materials in traditional Asian medicine for over two thousand years. With recent global warming and overharvest, however, the availability of these wild fungi has decreased dramatically. While fruiting body of O. sinensis has been artificially cultivated, the molecular mechanisms that govern the induction of fruiting body at the transcriptional and post-transcriptional levels are unclear. In this study, we carried out both mRNA and small RNA sequencing to identify crucial genes and miRNA-like RNAs (milRNAs) involved in the development of fruiting body. A total of 2875 differentially expressed genes (DEGs), and 71 differentially expressed milRNAs (DEMs) were identified among the mycoparasite complex, the sclerotium (ST) and the fruiting body stage. Functional enrichment and Gene Set Enrichment Analysis indicated that the ST had increased oxidative stress and energy metabolism and that mitogen-activated protein kinase signaling might induce the formation of fruiting body. Integrated analysis of DEGs and DEMs revealed that n_os_milR16, n_os_milR21, n_os_milR34, and n_os_milR90 could be candidate milRNAs that regulate the induction of fruiting body. This study provides transcriptome-wide insight into the molecular basis of fruiting body formation in O. Sinensis and identifies potential candidate genes for improving induction rate.

Overexpression of ribonucleotide reductase small subunit, RNRM, increases cordycepin biosynthesis in transformed Cordyceps militaris.

Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn't been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases (RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR (qRT-PCR) and western blotting results showed that the mRNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wild-type strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn't remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine (3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.

Comparative transcriptome analysis revealed genes involved in the fruiting body development of Ophiocordyceps sinensis.

Ophiocordyceps sinensis is a highly valued fungus that has been used as traditional Asian medicine. This fungus is one of the most important sources of income for the nomadic populations of the Tibetan Plateau. With global warming and excessive collection, the wild O. sinensis resources declined dramatically. The cultivation of O. sinensis hasn't been fully operational due to the unclear genetic basis of the fruiting body development. Here, our study conducted pairwise comparisons between transcriptomes acquired from different growth stages of O. sinensis including asexual mycelium (CM), developing fruiting body (DF) and mature fruiting body (FB). All RNA-Seq reads were aligned to the genome of O. sinensis CO18 prior to comparative analyses. Cluster analysis showed that the expression profiles of FB and DF were highly similar compared to CM. Alternative splicing analysis (AS) revealed that the stage-specific splicing genes may have important functions in the development of fruiting body. Functional enrichment analyses showed that differentially expressed genes (DEGs) were enriched in protein synthesis and baseline metabolism during fruiting body development, indicating that more protein and energy might be required for fruiting body development. In addition, some fruiting body development-associated genes impacted by ecological factors were up-regulated in FB samples, such as the nucleoside diphosphate kinase gene (ndk), ß subunit of the fatty acid synthase gene (cel-2) and the superoxide dismutase gene (sod). Moreover, the expression levels of several cytoskeletons genes were significantly altered during all these growth stages, suggesting that these genes play crucial roles in both vegetative growth and the fruiting body development. Quantitative PCR (qPCR) was used to validate the gene expression profile and the results supported the accuracy of the RNA-Seq and DEGs analysis. Our study offers a novel perspective to understand the underlying growth stage-specific molecular differences and the biology of O. sinensis fruiting body development.

[SSR loci information analysis in transcriptome of Cordyceps sinensis].

To analysis the SSR loci information in the transcriptome of Cordyceps sinensis and develop SSR molecular markers,MISA(MicroSatellite) software was used to analyze the microsatellites information from 16 875 unigene sequences and SSR primer designed by Primer 3. 0. In total,5 899 SSRs were detected in 4 252 unigene with the distribution frequency of 34. 99%,which was represented by 74 repeat motifs and SSR loci occurred per 7 952 bp in length. In the SSRs,the mono-nucleotide was the most abundant repeat motif(42. 5%),followed by tri-nucleotide(34. 48%),C/G and CCG/CGG were the dominant repeat motifs,respectively. The number of repetitions of the six SSR repeat types was concentrated on 5 to 12 times,and the length was mostly less than 24 bp. A total of 12 282 pairs of primers were screened and selected 20 pairs of primers for validity detection randomly,10 pairs of primers amplified the expected specific bands,and primer P1 has significant polymorphism. Moreover,it was found that unigene containing SSR loci is mainly related to genetic and environmental functions after GO and KEGG annotation. In conclusion,these SSR loci in the transcriptome of O. sinensis are high in frequency,rich in primitive types,high in polymorphism,and highly available,which will provides abundant candidate molecular markers for its genetic diversity analysis,resource identification protection,and gene function research.