Studies On Cordyceps Militaris Degeneration And Regulation Mechanism Of Cordycepin Metabolism

Cordyceps militaris is the model species of Cordyceps.With the rapid development of the cordyceps industry,the degeneration of Cordyceps militaris,the regulation of cordyceps metabolism,and the establishment of molecular breeding system have become the three major technical challenges in the production industry.In this study,based on the monoconidia strain determined by mating type gene as the research object,the mechanism and countermeasures of the degradation of Cordyceps militaris strain during the subculture process were discussed.The fruiting bodies with high cordycepin content were treated with different concentrations of naphthoacetic acid(NAA).The molecular mechanism of cordycepin synthesis was investigated.In order to provide ideas and clues for molecular breeding of Cordyceps militaris,the high-yield materials obtained by natural breeding were selected as the research object,and the differences between high-yield strains and wild strains were compared by resequencing method.1.Three producing strains and three wild-type strains of Cordyceps militaris were selected as the research objects,and the monoconidial strains of each strain were isolated and obtained.The effects of subculture of the original strain and the monoconidial strain on mushroom emergence were compared,and the changes of mating types of the original strain and the degenerate strain were detected.On this basis,two mating types of different proportions of inoculation experiments were designed to determine their effects on fruiting.Results The five original strains of Cordyceps militaris tested were heteronuclear strains(containing two mating type genes at the same time),while the production strain jb-2 was a mutant strain that could produce mushrooms with single mating type(MAT1-1)but could not produce sexual spores.In the process of subculture,the loss of certain mating types or the imbalance of mating types was the main cause of degeneration.The loss or imbalance of mating types of MAT1-1 and MAT1-2 was random.The subculture stability of different strains was significantly different.The single mating strain jb-2had strong stability in fruiting and could produce mushrooms normally after being transmitted to 11 generations.The production strain and the wild strain inoculated with different mating type ratio had different effects on mushroom emergence.The production strain inoculated with different mating type ratio of 1:9 to 9:1 did not affect the yield,and its stability was better.The yield of wild-type strain(xf-1)was positively correlated with the proportion of MAT1-2,while the other two strains had no correlation.2.The transcriptome and metabolome association analysis of Cordyceps militaris treated with different concentrations of NAA was carried out.The genes related to cordycepin synthesis and metabolites of purine metabolic pathway were significantly changed under each treatment concentration.Through the analysis of the regulatory network between genes and genes,as well as between genes and metabolites,a metabolic pathway map was proposed,including the joint action of key genes and metabolites in cordycepin synthesis,purine metabolism,TCA cycle,pentose phosphate pathway,alanine,aspartic acid and glutamic acid metabolism,and histidine metabolism.At the same time,the ABC transporter pathway was significantly enriched.It was found that the ABC transporter transported a large number of amino acids such as L-Glutamate,which participated in the amino acid metabolism affecting the synthesis of cordycepin.3.Tissue separation and stable fruiting experiments were conducted on the mutant fruiting bodies of a single wild Cordyceps militaris strain,and a high-yielding strain with a higher biological conversion efficiency than that of the wild strain was selected.Wild single mating strain and high yield single mating strain were tested to produce mushrooms,and single mating strain(MAT1-1 type)was identified as high yielding material.In order to track the causes of high yield,the high-yielding single mating strains and wild single mating strains were analyzed and compared by whole genome sequencing based on resequencing method.The results showed that the high yield single mating strain had more differential gene information than the wild single mating strain.In conclusion,this study laid a foundation for the research on the mechanism of Cordyceps militaris degeneration and high yield and good quality characters.The method of subculture of single conidia strains and repairing before production and use can effectively solve the degeneration problem in the subculture process.In the direction of Cordyceps militaris breeding,the strains with double mating type could be selected which were not sensitive to the proportion of mating type,and the strains with stable subculture and not easy to lose mating type.It is also an effective breeding route to select special strains of single mating type.High cordycepin strains were obtained by NAA treatment,and multiple pathways worked together to double the cordycepin content,which provided an important reference for the molecular network relationship between transcription and metabolism of cordycepin synthesis.The reason for the high yield of single mating strain(MAT1-1 type)needs further experimental verification.In the future,high yield single mating strain materials will be used to construct the hybrid population of Cordyceps militaris,and the extreme character analysis method of BSA will be used to further excavate the candidate gene design markers to establish the molecular breeding system of Cordyceps militaris.