Estabishment And Optimization Of Genetic Transfermation System For Streptomyces 769

This study was based on the determination of Inhibitory spectrum and growth curve, through the conjugation transfer and electroporation methods and optimized to establish genetic transformation system for streptomyces 769. Obtain the following results.1.Based on experiments in vitro, streptomyces 769 showed a broad-spectrum inhibitory activity against 16 species of plant pathogenic fungi with different sensitivities, such as Magonaporthe grisea, Exserohilum turcicum, Sphacelotheca cruenta, Gibberella zeae, Alternaria porri, Colletotrichum coccodes et. al.2.Liquid culture conditions of streptomyces 769 was screened, and its growth curve was determined by the nephelometery, dry weight of mycelium and bacteriostasis-diameter as well. The growth curve indicated that its lag phase, log phase, stationary phase, and decline phase were 0-36h,36-84h,84-108h, and 108h after inoculation in YEME medium under shaking cultivation, respectively.3.The conjugation transfer and electroporation methods were used and optimized to establish genetic transformation system for streptomyces 769. In this article, the genetic integration plasmid pSET152, ET12567 (pUZ8002, pSET152), and Streptomyces 769 spores were used as the starting plasmid, the donor, and the receptor, respectively. The results indicated that the pre-germination conditions of spores were heat shock at 50℃for 15 min in MS medium, with a ratio 10:1 of the donor strains to receptor strains,16 to 18 hours in antibiotics, the transformation efficiency was about 0.3×10-5. The anti-apramycin gene acc(3)Ⅳgene was transformed and integrated successfully into streptomyces 769 genome identified by PCR. Electro-transformation was also used for Streptomyces 769 genetic transformation system establishment. The transformation reaction was tested under different electric field strength. The results showed that the optimized conditions were in 106 spores/ml,2.5Kv electrical pulse intensity,3.1 milliseconds shock time,15ug/ml apramycin, respectively. Transformants were obtained and identified by PCR. However, we still need to pay more attention on improving the electro-transformation efficiency and transformants genetic stability in future.