Analysis Of Ergosterol And The Genes Associated With Its Synthesis In Cordvceros Spp.

Ergosterol is the main sterol component of Cordyceps fungi,which has anti-tumor,anti-inflammatory,anti-fibrosis and other pharmacological activity,and has shown great potential value in the field of medicine.However,the systematic exploration and evaluation study on the ergosterol of Cordyceps has been lacking,and the basic research and development of ergosterol in Cordyceps has been still weak.In the present study,the extraction methods,content distribution and metabolic regulation genes of ergosterol were studied in Cordyceps spp.The main results were as follows.1 The method of response surface analysis was used to optimize the extraction method of HPLC determine Cordyceps ergosterol.The solvent concentration,solid-liquid ratio,soaking time,ultrasonic time and ultrasonic numbers were taken as the preliminary factors to evaluate the single factor tests.On this basis,the effects of solid-liquid ratio,soaking time and ultrasonic time on the extraction of Cordyceps ergosterol were studied by response surface analysis method.The optimum extraction conditions obtained in this experiment were as follows:extraction solvent 100%methanol,solid-liquid ratio 1:40,soaking time 3.3 h,ultrasound time 50 min,ultrasonic extraction twice.Three replicated experiments were carried out to verify the predicted values.Combining with the method validation,it was proved that the extraction and determination method were stable and feasible.2 The biomass and ergosterol content of eight Cordyceps mycelial were determined during the culture time.Combined with the total ergosterol content which was the comprehensive value of biomass and ergosterol content,the time curves were drawn to analyze the dynamics changes of the three in the cultivation.And the correlation between ergosterol content and biomass of Cordyceps mycelium was analyzed.The results showed that the changes of biomass mostly increased firstly and then flattened and declined at last,while the ergosterol content continuously increased with the culture time.The correlation analysis showed that there was no correlation between the biomass and ergosterol content in the whole growth process,but there was a positive correlation between the two in the biomass maximum prophase period.This showed that the ergosterol content indicated the biomass did not apply to the whole growth period of Cordyceps.Meanwhile the results showed that the ergosterol content had no definite maximum harvest period in 100 days.3 The ergosterol content of Cordyceps mycelium was determined by using 70 strains of 7 species(10 strains per species)in its biomass maximum period.The distribution of ergosterol content in different strains and species was analyzed,and high yield strains were screened according to ergosterol content and biomass as well.It was showed that biomass and ergosterol content had a great discrepancy in different strains,especially ergosterol content might range from 0.34 mg/g to 12.22 mg/g.While there was no significant difference between species.In the nested analysis,the variations in ergosterol content and total ergosterol content were mainly from the strains(variation percentages were 92.55%and 94.28%,respectively),while the variations in biomass were mainly from the species(variation percentage was 59.06%).Meanwhile,the biomass and ergosterol content distribution in the same strain was inconsistent.We screened strain OLP104XS10 with high total ergosterol content(9.98 mg/100mL)which could be used as a good production strain.4 The 23 genes of ergosterol synthesis pathway were discovered and indentified in Saccharomyces cerevisiae.In the basis,the metheod of homologous BLAST was used to achieve the ergosterol metabolic pathway genes of Cordyceps in the GenBank database.And the bioinformatics technique was used to analyze the sequence characteristics of Cordyceps metabolic genes.Four sequence primers were designed to amplify the four key genes(ERG4,ERG6,ERG7,ERG9)of Cordyceps,and the genetic variation and evolution of ergosterol metabolism genes in Cordyceps were analyzed.The 22 metabolic gene sequences were obtained from 15 Cordyceps species which had been completed whole genome sequencing successfully.And the database of ergosterol metabolic pathway was established.The multi-gene cluster tree analysis showed that the genetic distance between different species was obvious,and the topology was consistent with the phylogenetic system,indicating that it could be a good material for phylogenetic development.The gene sequences of ERG4,ERG6,ERG7 and ERG9 were obtained by gene amplification of 19 isolates of ergosterol content strains of each 7 Cordyceps species.Single gene system trees and mutation sites showed that the variation of the sequence of different species was different.In the same species,there were rich mutations in some species except for Isaria cicadae,but we did not find the correlation between variation of single nucleotide and amino acid and the difference of ergosterol content in Cordyceps.In conclusion,the results of this study provided a quick and effective method for the determination and quality control of Cordyceps ergosterol.The ergosterol content changes during the cultivation of Cordyceps mycelium were verified,suggesting that ergosterol content as the indicator of Cordyceps biomass had a certain time limit.The distribution characteristics of ergosterol in Cordyceps species and their strains were also discussed,which provided a theoretical basis for screening and application of high yield strains.The gene analysis of ergosterol metabolic pathway in Cordyceps was carried out to provide some basic theoretical basis for further excavation of drug targets and genetic engineering breed.