| Ophiocordyceps sinensis (Berk.) GH Sung et al.[=Cordyceps sinensis (Berk.) Sacc, anamorph:Hirsutella sinensis Liu, Guo, Yu & Zeng] (Ascomycota) parasitizes approximately 57 different species of caterpillar larvae belonging to the family Hepialidae (Lepidoptera). Chinese caterpillar fungus, a "treasure of tonics", along with ginseng and deer antler, has been used for thousands of years in Asian medicine. The complex of the mummified caterpillar and the fungal stroma resulting from parasitism is renowned and widely used for the treatment of asthma, bronchitis, lung inflammation, nocturnal emissions, night sweats, and other diseases. The natural distribution of O. sinensis and its host is limited to Qinghai-Tibetan Plateau, between 3,000 and 5,000 m elevation, including parts of China, Bhutan and Nepal. Due to its famous curative effect and reduced supply, the current price of O. sinensis has reached $ 53.7 per gram, causing it to be considered as "golden" Chinese medicine. O. sinensis has been listed as an endangered species of national key preserved wild plants. The high price has led to the appearance of many counterfeited products on the market, which have morphological characteristics that are barely distinguished based on morphology alone, thereby causing confusion and threatening its safe use. The fruiting body of O. sinensis is the primary part used for identification and medicinal purposes. However, the fruiting body of O. sinensis is hard to culture in vitro, and the sexual and fruiting body development of O. sinensis is unclear. At present, the genomic and transcriptomic data are still unavailable in the GenBank database. To protect wild resource, strengthen market management and promote researches in molecular biology, modern technologies, including remote sensing (RS), traditional Chinese medicine geographic information system (TCMGIS), DNA barcode and high-throughput transcriptome sequencing, were used in this study.A long time-series (8 km resolution) NDVI dataset was used to study the dynamic changes in the vegetation cover in four major production areas of Chinese caterpillar fungus during 1981～2010. As quantified by remote sensing, the results indicated that the habitat availability of O. sinensis has been decreased during 1981～ 2010. Furthermore, TCMGIS was used to analyse optimal ecological factors and regional distribution. The optimal ecological factors for the herb are mean annual temperature 4.3～16.9℃; mean temperature in January-16.9～2.6℃; mean temperature in July 5.0～17.0℃; annual sunshine 1,666～2,943 h; annual precipitation 363～922 mm; relative humidity 52.6%～71.9%. The appropriate district (similarity≥95%) for the O. sinensis is 726,917.7 km2 in 205 counties in the southeast of Qinghai-Tibet Plateau. Those counties are ideal areas chosen for artificial cultivation and wildlife tending for O. sinensis.Artificial cultivation or introduction technology may be the most effective tool to meet the market demand and alleviate the pressure to overexploit wild O. sinensis. Based on the optimal ecological factors, Kangting county in Sichuan province, which is one of the producing areas, was chosen to cultivate O. sinensis. A total of 3,035 O. sinensis individuals were cultured in an artificial environment, and the production cycles of O. sinensis can be decreased to one year instead of 4-5 years in the wild. We also identified an excellent host (Hepialus xiaojinensis), which was perfect for captive breeding in artificial environment because the caterpillar grows rapidly and exhibits a strong resistance to disease. H. xiaojinensis also displays a high reproduction rate which would be beneficial for re-introducing population to their native habitat. However, the artificial cultivation of O. sinensis still had the associated problems of high cost and low production efficiency, which should be addressed for viable commercial production.Due to the tremendous demand, the limited wild resources, and rare commercially available products from artificial cultivation, various counterfeit versions of O. sinensis resulted in confusion in the market and threatened drug safety. To verify the applicability of DNA barcoding, ITS sequence has been used for fast and accurate identification of 131 O. sinensis samples and 13 common counterfeits or closely related species. A total of 18 new ITS haplotypes of O. sinensis and counterfeits or closely related species generated in this study were submitted to GenBank. The polymerase chain reaction amplification rate of six Ophiocordyceps species was 100%. The distinct barcode gaps in six Ophiocordyceps species clearly represented each of these closely related species. BLAST1 and Nearest genetic distance method revealed identification efficiencies of 100%. The ITS haplotypes of the species used in this study formed distinct, non-overlapping monophyletic groups in the Neighbor-Joining (NJ) tree, Maximum Parsimony (MP) tree, Maximum Likelihood (ML) tree. These results indicated that DNA barcoding could be used as a fast and accurate identification method to distinguish O. sinensis from counterfeits or closely related species for its safe use.Furthermore, Roche 454 technology was used to analyze the transcripts of the O. sinensis fruiting body. A total of 34,289 unique sequences were obtained, including 17,059 contigs and 17,230 singletons, for the study of the developmental biology of the fruiting body and for the discovery of genes involved in cordycepin biosynthesis in this traditional medicinal fungus. Based on the GO and KEGG analysis, the expression patterns and function of these genes were illustrated. Most of the genes involved in sexual and fruiting body development, including the mating type locus, genes involved in the signal transduction pathways and transcription factors, were discovered in this 454 dataset. This is the first report of three MAT1-1 genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) in O. sinensis, and all the genes involved in both the PKA and MAPK signaling pathways were found. Additionally, the transcription factors related to fruiting body development were abundant, including C2H2, Zn2Cys6, and bZIP. These results indicated that the sexual and fruiting body development in O. sinensis may be different from those in C. militaris. Furthermore, all the enzymes involved in the putative biosynthesis pathway of cordycepin were discovered in O. sinensis. Additionally,1,255 simple aequence repeats (SSRs) were searched in 1,235 unique sequences, most of which were trinucleotide loci (58.65%). Based on this transcriptome dataset, more special genes and genetic knowledge of O. sinensis will be understood in the future.In summary, the environment of O. sinensis will be effectively monitored based on the remote sensing. TCMGIS and artificial cultivation will alleviate the pressure to overexploit wild O. sinensis. DNA barcoding can be taken to manage collection and trade.454-EST transcriptome dataset will further study the molecular biology in O. sinensis. This integrated strategy will contribute to resources protection and sustainable utilization for O. sinensis... |
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