Transcriptome Study Of Different Morphology Of Mycelium Before Sexual Development Of Ophiocordyceps Sinensis

This paper summaries the geographical distribution,morphological characteristics,life history,host insects,important active components and the asexual type of Cordyceps sinensis.The morphology of mycelium and genes related to fruiting body of edible and medicinal fungi were reviewed.The asexual type of Cordyceps sinensis has always been controversal.However,a variety of mycelium type appeared in the culture of Ophiocordyceps sinensis under laboratory conditions,it may be due to the presence of other bacteria.In addition,there is a lack of systematic research on the phenomenon of pleomorphism in the mycelium of Ophiocordyceps sinensis.Therefore,in order to investigate the difference and causes of the three morphology of mycelia(aerial mycelium,hyphae knot and substrate mycelium)that appeared in the artificial culture of Ophiocordyceps sinensis,a strain isolated from single ascospore as materials the microstructure and ultramicrostructure of the three morphology of mycelia were observed with microscopy and scanning electron microscopy respectively.At the same time,a preliminary study on transcriptomics was carried out.The main results are as follows:1.Characteristics of strains.Three kinds of mycelia appeared in the culture of the isolates of Ophiocordyceps sinensis,which derived from a single ascospore and obtained by tissue isolation.The results showed that three morphology mycelia of Ophiocordyceps sinensis are the normal characteristics of the species and are not impacted by the purity of the strain or species,which was one of the characteristics of the strain.2.Observation of ultramicroscopic characteristics of three morphology of mycelium for Ophiocordyceps sinensis.Three morphologies of mycelia in the artificial culture of Ophiocordyceps sinensis were observed with microscope and scanning electron microscope(SEM).The results showed that the diameter of the hyphae were manifested as the hyphae knot>aerial mycelium>substrate mycelium,the aerial mycelia were loosely arranged and attached with a large number of conidia on the surface of mycelia.The hyphae knot had obvious fold,kink and cross-linked between each other.The substrate mycelium was densely arranged,and the hyphae fused and branched obviously.A large number of conidia were observed and attached to the aerial hyphae,which appeared in the late culture period of Ophiocordyceps sinensis,and the conidia were born directly by budding at the end or middle of aerial hyphae,indicating that the way of producing conidia in the aerial hyphae was budding.3.Transcriptome sequencing.Three kinds of mycelia were taken for transcriptome sequencing and De Novo assembly.The 13,374 Unigene were obtained.A total of 921differentially expressed genes(DEGs)were found in hyphae knot vs substrate mycelium,among which 593 genes were up-regulated and 328 genes were down-regulated.KEGG enrichment results showed that up-regulated DEGs were mainly concentrated in the oxidative phosphorylation metabolic pathway,providing energy for the formation of hyphae knot.There were 1827 DEGs in aerial mycelium,926 genes were up-regulated and 901 genes were down-regulated.The up-regulated DEGs were mainly enriched in the pathways of Valine,leucine and isoleucine biosynthesis,suggesting that the synthesis of branchy-chain amino acids provided nitrogen sources for the development of aerial mycelium in the late culture period.There were 1471 DEGs in aerial mycelia and hyphae knot,of which 709 were up-regulated and 762 were down-regulated.The up-regulated differential genes were enriched in ribosomes and peroxidasomes in the substrate mycelium,indicating that nutrition was abundant at the early stage of culture and metabolism was vigorous in the cells of the substrate mycelium.The reliability of the transcriptome data was verified by q RT-PCR.4.Study on reference genes.18S r RNA,APRTase,?-TUB,RPL2,EF1-?,PGI,PGM,H~+-ATPase,ACT1,UBQ and GAPDH were selected as candidate reference genes.The primers were designed and amplified by q RT-PCR.The expression of stability was evaluated by ge Norm,Norm Finder and Best Keeper algorithms,and the evaluation of results were combined Ref Finder to screen the optimal internal reference genes.The results showed that all the 11 candidate reference genes could be used as reference genes in the mycelium stage of Ophiocordyceps sinensis,the most stable were UBQ,PGM and ACT1.