| Cordyceps is one of the most special fungi resources in China while its artificial cultivation,pharmacodynamic components and pharmacological effects have attracted the attention of scholars.Ophiocordyceps xuefengensis is a new kind of cordyceps resource discovered in Xuefeng Mountains,Hunan Province,in recent years.It has a close relationship with O.sinensis and was used in Dongkou County for the treatment of cancer,cough,and immunity.However,the phylogenetic relationship of O.xuefengensis is unclear,and wild resources are scarce,so artificial cultivation needs to be studied.This study investigated O.xuefengensis resources and ecological environment,obtained the mitochondrial genome of O.xuefengensis through three-generation sequencing to study its systematic evolutionary relationship.Successfully cultivated O.xuefengensis complex and artificial progeny,and found that light has a significant effect on its growth and development.The key influence is to further study its light quality effect,and obtain biological information of WCC,the only photoreceptor system of O.xuefengensis from the transcription data,and cloned and analyzed its photoreceptor gene.The specific results are as follows:1、O.xuefengensis mostly grows in the roots or stems of the living plant C.cyrtophyllum at an altitude of 300-1 000 m.Dongkou County is the core distribution area.The average length of the larvae is 7.72 cm and the average diameter is 7.90 mm.The stromata were mostly solitary from the head of the larva,yellow-brown,cylindrical,upright or curved,with an average length of 9.34 cm and an average diameter of 2.39 mm.The top part branches and develops into a fertile part with ascus shell.The analysis of the microbial community of the wild O.xuefengensis found that O.xuefengensis had the highest abundance,followed by Beauveria bassiana and Metarhizium anisopliae,and Fusarium was also found.Ten strains were isolated from the wild O.xuefengensis,and identied by the ITS sequence.2、In this study,we sequenced,assembled,and annotated the entire mitochondrial genome of O.xuefengensis,in addition to comparing it against other three complete cordyceps mitogenomes that were previously published.Comparative analysis indicated that the four complete mitogenomes are all composed of circular DNA molecules,although their sizes significantly differ due to high variability in intron and intergenic region sizes in the O.sinensis and O.xuefengensis mitogenomes.All mitogenomes contain 14 conserved genes and two ribosomal RNA genes,but varying numbers of t RNA introns.The Ka/Ks ratios for all 14 PCGs and rps3 were all less than 1,indicating that these genes have been subject to purifying selection.Phylogenetic analysis was conducted using concatenated amino acid and nucleotide sequences of the 14 PCGs and rps3 using two different methods(Maximum Likelihood and Bayesian analysis),revealing highly supported relationships between O.xuefengensis and other Ophiocordyceps species,in addition to a close relationship with O.sinensis.These mitogenomes will allow further study of the population genetics,taxonomy,and evolutionary biology of medicinally important cordyceps species.3、The suitable temperature of O.xuefengensis fermentation growth is 25 ℃,and the biomass of O.xuefengensis fermentation broth is significantly higher under breathable than that under airtight conditions.Through fermentation kinetics research,it was found that the fermentation mycelial biomass reached the maximum(15 g/L)in about 30 d.The method of cultivation of cordyceps complex is to select a big larva,disinfect with 75% alcohol and UV for 20 min,and then inject 0.2～0.3 ml of mycelium suspension at a temperature of 7～10 ℃.Transferred the larvae to 20～25 ℃ when there is a substratum primordium on the body surface.The optimal medium for artificial stromata culture is 15 g rice + 2 g silkworm pupa powder + 60 m L liquid medium.The pedestal has obvious phototropism.It is speculated that light may be a key factor affecting the formation and growth of the stromata.4、Light is a critical environmental factor for the stromata formation and development of many fungi.In this study found that light has no effect on the growth rate of the colony,but it has a greater influence on the morphology and color of the colony.And the light intensity greater than 50 lux obviously inhibits the formation and growth of stromata.And light represses vegetative growth,but conidia linked to stromata can be observed under both light and dark conditions.Light and dark conditions conducive to the accumulation of carotenoids,and activates SOD and POD enzyme activities to protect the body,but had little effect on the accumulation of polysaccharides and adenosine.5、Photoreceptors are proteins that receive light signals.We found the only photosensitive system WCC by analyzing the transcriptome data of O.xuefengensis under light and dark conditions.The genes Oxwc-1 and Oxwc-2 encoding photoreceptors of O.xuefengensis were cloned and predicted to possess polypeptides of 937 and 525 amino acids,respectively.A phylogenetic analysis based on fungal WC-1/2 supported Ox WC-1 and Ox WC-2 were photoreceptor.The expression of both the Oxwc-1 and Oxwc-2 genes reached a maximum after receiving light stimulation for 15 min,which might relate to the inhibition of stromata growth. |
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