| Pleurotus ostreatus is one of the earliest domesticated edible mushroom species,with the second-largest production in the world,and China is the largest producer and consumer.The fruiting bodies are nutrient-rich,with a balanced amino acid ratio,and the polysaccharides have a positive effect on inhibiting tumor growth and enhancing immunity.The taste and nutritional content of the caps are higher than the stipe,and the short-stipe phenotype is more popular in the consumer market,but it is difficult to control the developmental direction of the fruiting body due to facility conditions and cultivation costs.The subculture of strains in production is a widely used method of preservation and breeding,subculture will cause strain degeneration,due to the unclear molecular mechanism of degeneration and the lack of detection and predictive means,the degeneration phenotype will only show up in the development stage of the fruiting body,causing irreversible economic losses.In this paper,by analyzing the relationship between the stability of P.ostreatus P16 after continuous subculture and the proteome and genome changes,the molecular mechanism of strain degeneration was investigated,and the detection index was put forward.The differences between the stipe tissue and the cap tissue in the fruiting bodies were quantitative analyzed,exogenous inducers were screened to promote short-stipe phenotypes and increase the conversion efficiency and the commercial rate.The main research of the paper is divided into three parts:(1)After continuous subculture to P5,P10,and P15 generations of P.ostreatus,there was no significant difference in mycelial growth rate and synchronization.The converted efficiency of P10 was 21.3%lower than that of P1,and the converted efficiency of commercial mushroom was reduced by 30%(p<0.01).Label-free proteomics was used to compare the differences in protein expression between P10 and P1 strains.The activity of hydrolase was significantly changed after subculture(p<0.001).It was found that after subculture,cell cycle arrest,base excision repair,non-homologous end-joining pathway-related proteins expression were up-regulated,homologous recombination related proteins were down-regulated.According to the quantitative results of protein expression in proteomics and the enzyme activity data of mycelium,it was determined that the activities of laccase,α-galactosidase,carboxylate hydrolase,and catalase were strongly correlated with the converted efficiency of the fruiting body(p<0.001),and Pearson correlation coefficients were-0.8674,-0.8702,-0.9576 and 0.7837.When the enzyme activity of the 4 index enzymes changed significantly at the same time(p<0.05),the strain was judged to be degraded.(2)The genome sequence and expression regulation of strains in the succeeding culture process were found to occur by examining the changes in esterase isozyme profile and DNA polymorphism.DNA methylation plays an important role in the maintenance of genetic material stability and regulation of gene expression in the organism.P1,P5,P10,and P15 genomic methylation levels were 1.76%,2.02%,2.45%,and 2.79%,quantitative analysis showed that the genomic methylation level increased significantly with the increase of the number of successive cultures(p<0.01)The results of genome-wide methylation sequencing showed that the methylation levels of genes related to synthesis and metabolism,repetitive element maintenance,cell cycle,external stimulus-response and homeostasis of the internal environment were increased(p<0.01),while methyltransferase activity,non-homologous end-joining,protein extracellular transport,negative regulation,and galactose metabolism-related genes were decreased(p<0.01).The results of proteome and genome co-analysis showed that genome damage caused by endogenous factors inducing DNA double-strand breaks was the fundamental cause of strain degeneration in the subculture.Non-homologous end-joining repair led to genome homeostasis disorder,which promoted strain degeneration,while DNA methylation accelerated the process.(3)The iTRAQ proteomics was used to compare the protein expression in the stipe and cap tissues.The results showed that there were significant differences in sphingolipid metabolism,starch and sucrose metabolism,sphingolipid signaling pathway,steroid biosynthesis,and bicarbonate reabsorption pathway(p<0.01).Real-time quantitative PCR analysis of gene relative expression confirmed the accuracy of the proteomic results.The metal ion concentration determination results showed that the Ca2+ and Fe3+ content in the cap was significantly higher than that in the stalk(p<0.05).According to the results of proteome and ion concentration determination.CaCl2.ergosterol,NaHCO3.and other inducers were prepared.The results of the exogenous induction experiment showed that the average length of stipe was shortened by 20.36%and the average yield of the fruiting body was increased by 15.27%after continuous treatment with 1 mM CaCl2(p<0.05).Under high CO2 concentration stress or high-temperature stress,continuous spraying of CaCl2 at the primordial stage could significantly increase the commercial rate of fruiting bodies by 14.04%and 22.30%.The results showed that exogenous Ca2+ played a key role in regulating the differentiation direction of fruiting bodies through induction and inhibition experiments,and sphingolipid was an important signal medium for regulating the differentiation of stipe and pileus.In summary,by tracking and analyzing the changes in the stability of successive strains,it is proposed that the reference index for determining the degeneration of strains can provide the theoretical basis and technical support for controlling the quality of spawns.By analyzing the difference between the stipe and cap of the fruiting body,the exogenous inducers were screened to achieve high quality and efficient cultivation of the P.ostreatus. |
PDF Full Text Request