Isolation And Identification Of Three Actinomyces Strains And Construction And Optimization Of Their Genetic Transformation System

The soil-dwelling streptomyces are major source of natural antibiotics.Isolation and characterization,establishment of genetic transformation system and the study on the function of several transcriptional of the soil-dwelling streptomyces not only benefit the exploration of the unique microorganism resources and the potential of biosynthesis of the new species but also provide the basis for analysis the function of the vitial gene clusters and develop necessary drugs in the fields of medicine and agriculture.In this study,we isolated 63 stains of actinomyces from the soils of QingShan Forest Farm of ChiFeng city in Inner Mongolia.Thought conducting bioactivity detection experiment on 63 strains respectively,We obtained 7 stains of actinomyces which had better actibacterial activity,and then,constructed the genetic transformation system for 7 stains of them.Finally,3 stains of those actinomyces was successfully established.S-5,S-27 and S-28,whose genetic transformation system had already constructed,were identified by morphological,cultural characteristics,physiological and biochemical characteristics with 16 S rDNA gene analysis.We came to the conclusion that 3 stains of Streptomyces were Streptomyces alogtiseolus,Streptomyces albus and Streptomyces bacillaris,respectively.The genetic transformation system of 3 strains of Streptomyces S-5,S-27 and S-28 was constructed by conjugation,and all of them of the genetic transformation system were constructed successfully.We optimized genetic transformation system of S-28,including the selection of culture,the concentration of MgCl2,Heat-shock temperature,inclubation time,Donor-to-Recipient Ratio,and so on.In conclusion,we got the optimal conditions.Whole-genome sequencing and bioinformation analysis showed that the genome size of strain S-28 was 7,864,237 bp,in which there were 91 scaffolds and 93 contigs,and GC content of it was 71.95%.It contained 6,882 coding genes which predicted by software.Combining the annotation of a variety of databases with analysis by antiSMASH,there were38 gene clusters,it showed that the strain contained abundant secondary metabolites resources,which had great value of development and utilization.Disrupting bldA and MarR by gene replaced and in-frame deleted.Heterologous expression of MarR transcription factor was conducted.Result: The MarR transcription factor was successfully expressed.The re-combinant plasmid pKC1132-L+Kan+R was transtformed into Streptomyces bacillaris by conjugation.After antibiotics resistance screening and PCR identification,but it was not confirmed that bldA was replaced in Streptomyces bacillaris S-28;Then the gene knock-out plasmid pKC1139-L+Kan+R was transformed into Streptomyces bacillaris with the same method,but MarR in-frame deletion strain Streptomyces bacillaris S-28 was not obtained.