The Mechanism Of Methylation-related Genes Inthe Degradation Of Cordyceps Militaris

Cordyceps militaris is an important fungus for food and medicine,and the technology for its artificial cultivation and production is becoming increasingly sophisticated.However,the frequent degradation of the strain in its conservation and production has greatly limited the quality development of the Cordyceps militaris industry.In this study,the normal strain Cm HY-1(double mating type)of Cordyceps militaris was cultivated and the adenosine and cordycepin contents of strains Cm HY-1,Cm HY-7 and Cm HY-9 in liquid culture were measured by succession culture,and strains Cm HY-1,Cm HY-5,Cm HY-7 and Cm HY-9 were prepared respectively.The methylation-related gene expression of strains Cm HY-1,Cm HY-7 and Cm HY-9 was measured.The degenerate strain Cm HY-7(mating type MAT1-2)was used as the starting strain to further construct the methylation-related gene overexpression strain,and the mycelial growth rate and conidial production were measured.The methylation-related genes and mating genes were used to study the role of methylation-related genes and mating genes in the degeneration process of Cordyceps militaris.The results showed that the strain degenerated during the succession culture compared with the normal strain Cm HY-1.The content of adenosine and cordycepin in strain Cm HY-9 decreased significantly,and the proportion of single-spore isolates with the mating type MAT1-1 decreased.The expression of methylation-related genes in the degenerated strains changed more significantly,with Cm Hln,Cm Dmta and Cm Sam genes decreasing significantly in Cm HY-9,while the expression of Cm Dim-2,Cm Hh3,Cm Hmd,Cm M16,Cm Lsd,Cm Hn and Cm Mfl genes increased extremely significantly.The colony diameters of the Cm HY-7 overexpression strain Cm MFT increased significantly,while the colony diameters of the overexpression strains Cm DMTA,Cm HH3,Cm HMD,Cm M16,Cm LSD,Cm HN and Cm MFL all decreased significantly compared to the control.The degenerate strain Cm HY-7,mating type MAT1-2,was unable to form substrates,but it was still invasive to the pupae,and most of the methylation-related gene overexpression strains constructed from it also failed to form substrates;co-cultures of Cm HY-7 with the normal Cm HY-1subcultural isolate(mating type MAT1-1)still failed to grow substrates.However,most of the overexpression strains were able to form small substrates after co-cultivation with the normal strain Cm HY-1,especially the overexpression strain Cm HN,which formed complete substrates(with ascospores).From the above results,it can be speculated that mating-type genes and methylation-related genes may play a synergistic role in the degeneration of Cordyceps militaris.This study also explored the genetic evolutionary lineage,physicochemical properties,conserved and functional domains,as well as the three-dimensional structure and interacting proteins of the proteins encoded by methylation-related genes in Chrysalis through bioinformatics analysis.Meanwhile,the overexpression of methylation-related genes and knockdown of key genes were carried out using Cordyceps militaris strain CM01(MAT1-1 monocross type),and phenotypic experiments were performed on the overexpressed and knockout strains.The results showed that,except for the gene overexpression strain CMHH3,the growth diameter of 21 d of the overexpression strain was significantly increased compared with CM01;the mycelium of the overexpression strains CM01 HLN and CM01HH3 grew twisted;the mycelium morphology of the other overexpression strains was consistent with CM01,and the mycelium tip of the knockout strain CM01-HN was completely curled and could not grow normally;the insectoid cultivation of the overexpression strains All of the overexpression strains could form complete zygotes,but the knockout strain CM01-HN could not form fruiting bodies on the worm body,while the other knockout strains could form fruiting bodies normally.