Genes Analyses And Identification Of Active Substance Produced By Streptomyces Tremellae

Present investigation was aim to study an anti-Mycogone perniciosa substance purified from the Js-1T).The active substance was analysed and proved to be H2Phe.The H2Phe bioactivity for Trichophyton mentagrophytes and HepG2 cell has been tested in this study.The whole-genome of Streptomyces Js-1T and cluster genes of H2Phe metabolism were also analysed in this paper.The active substance was purified by steps from concentration,ethanol removal impurity,n-butyl alcohol extraction,silica gel separation and HPLC purification.Mycogone perniciosa was used as the indicator on these processes.The optimized purification as follows:pH of extraction liquid should be between 6 and 7;the best mobile phrase of silica gel is chloroform:methanol:acetic acid=4:1:0.5;the active substance should be detected by HPLC with the condition of 20%methanol and its peak appeared on the time of 8.521min,the active substance should be purified by HPLC with the condition of 15%methanol and its peak appeared on the time of 10.259min.The active substance was detected by HPLC,ESI-MS and NMR and proved to be H2Phe,which molecular formula is C9H13NO2.The formula relationship between H2Phe concentration and peak area in HPLC is y=4000000x+86119 and R2=0.9973;The formula relationship between Phe concentration and peak area in HPLC is y=23758x+1402.8 and R2=0.99973.The formula relationship between H2Phe concentration and the rate of Mycogone perniciosa inhibition is y=0.18284x+0.00613 and R2=0.99925,so the minimal bactericidal concentration of H2Phe is 5.44mg/ml.H2Phe has no affect on rabbit unknown pathogens.The inhibition rate is up to 40%when the HepG2 cell were treated in 450μg/ml H2Phe at the time of 24h.When the concentration of H2Phe was over to 50μg/ml,the HepG2 cell has been high inhibited and there is no different inhibition rate for different H2Phe concentrations.When the concentration of H2Phe was over to 25μg/ml,it showed a high inhibition to the HepG2 cell in the research of clone-forming experiment.The sequence data of Streptomyces Js-1T whole-genome was 2.4GB and the splicing data of the whole-genome was 11.24Mb,so the coverage of sequencing was 219 times.The whole-genome splicing produced 7701 contigs,which average length is 1526bp and N50=7555bp.There are 121 Scaffolds,which average length is 96290bp and N50=229970,N90=49859,produced in the whole-genome splicing.The whole-genome were predicted to produce 11074 genes,all of the genes contain 10198965bp,which account 86.76%for the whole-genome.The number of CDS are 10953,repeat zone are 7 and the whole-RNA are 12.It has been predicted that PROKKA 00053 gene can produce the protein of PDXs and the metabolic pathway genes of H2Phe are surround beside on the PROKKA₀0053 gene.