Study On The Degradative Mechanism Of Cordyceps Militaris In Subculture

Modern medicine shows that Cordyceps militaris has various active substances,including polysaccharide,cordycepin,adenosine,cordycepic acid,ergosterol et al.Cordyceps militaris has remarkable effects on antioxidant,antibacterial,anti-virus and improving immunity,which is as a good substitute for expensive traditional Chinese medicine Cordyceps sinensis for its similar contents.Large scale artificial cultivation of Cordyceps militaris has been realized to relieve the tension of market supply of Cordyceps sinuses products.However,Cordyceps militaris would be degraded during the subculture,which significantly affected the development of fruiting body and hindered the efficient,large-scale and industrialized production of Cordyceps militaris.In this study,6 generations of Cordyceps militaris(Respectively named: YCCZ1-6)were cultured on the rice medium from the mother strain Cordyceps militaris strain YCCZ.The degradation and generation number were determined by fruiting body formation assay and mating type analysis,and then we analyzed the contents of active substances;finally,we analysised AS(Alternative splicing),DEGs(differentially expressed genes),GO(Gene Ontology)and KEGG(Kyoto encyclopedia of genes and genomes)pathway by transcriptional of 6 generation strains to explain the degradation mechanism of the Cordyceps militaris.1.The subculture and degradation analysis of Cordyceps militarisIn this study,we obtained 6 generations Cordyceps militaris strains by subculture.The fruiting body formation assay showed that the fruiting body of YCCZ1 and YCCZ2 appeared similar with orange color and normal shape;YCCZ3 had a light orange color;furthermore,YCCZ4 was short and thin;YCCZ5 and YCCZ6 failed to develop fruiting body;suggested the degradation phenomenon occurred from YCCZ3.The mating type of Cordyceps militaris strain is an important index to determine whether the strain is degraded.Therefore,we extracted the genome DNA of the 6 generation sub-cultured Cordyceps militaris and detected the mating type genes,MAT1-1-1,MAT1-1-2 and MAT1-2-1,by Touch-down PCR method.MAT1-1-2 fragment was deleted in YCCZ5 and YCCZ6 and The strain was degraded during the YCCZ3 and YCCZ4.2.The contents of active substances in mycelia of different generations of Cordyceps militarisThe degradation of Cordyceps militaris would directly affect the fruiting body formation,but also result in the contents of active substances.The content of Cordyceps militaris polysaccharide,ergosterol,cordycepin/adenosine,cordycepic acid was important detection index to measure the quality of Cordyceps militaris.Therefore,we analyzed the changes of4 kinds of active substances in mycelia by HPLC and colorimetric method.The results showed that contents of polysaccharide in YCCZ1-YCCZ3 were increased with the generations and reached the peak 54.55mg/g at YCCZ3;however,the contents of polysaccharide was plunged to 23.93 mg/g at YCCZ4,YCCZ5 and YCCZ6.The contents of ergosterol was in negative correlation with generations,which was decreased from 8.02mg/g in YCCZ1 to 0.77mg/g in YCCZ3.Contents of cordycepin and homologous adenosine were all in negative correlation with generation,comparing with YCCZ1,cordycepin was decreased 97.96% and adenosine 88.29%.Besides,the changes of cordycepic acid contents in 6 Cordyceps militaris strains were not significant,which was basically maintained at1.2mg/g.The results suggested that the fruit development of Cordyceps militaris significantly decreased the contents of polysaccharides,ergosterol and cordycepin/adenosine.3.Transcriptome analyses of different generations of Cordyceps militaris strainsWe extracted the total RNA of the 6 generation subcultures of Cordyceps militaris strains and performed the high flux transcriptional sequence assay to further explain the molecular mechanism of the degradation of Cordyceps militaris.Over 9,015 unigenes and 731 new genes were identified through transcriptional analysis.In addition,35,323 alternative splicing events were detected in all samples,and more AS events occurred in the second,fourth and sixth generations.Compared with the first generation,the third generation(degenerated strain)included 2,498 differentially expressed genes including 1,729up-regulated and 769 down-regulated genes.51 DEGs associated with strain degradation were validated through GO,KEGG and q RT-PCR method,which were involved in toxin biosynthesis,energy metabolism,DNA methylation and chromosome remodeling.