Interspecific Protoplast Fusion Between Streptomyces Roseoflavus Men-myco-93-63 And Streptomyces Lividans TK24

Streptomyces roseoflavus Men-myco-93-63 could inhibit many pathogen fungus and bacteria in lab. The fermentation liquid had a strong antibiosis activity against cotton verticillium wilt and cucurbits powdery mildew in greenhouse and field. The effective elements and function mechanism were under study. Parental strains of inactivated protoplasts fusion can get rid of the obstacles that genetic markers brought to breeding work, make the screening of fusants intuitive, and it is a good mearure of breeding high-yeilding strains and to be popularized.The research was carried out to inactivate protoplasts of Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24 using heat and UV, respectively, and two inactivated protoplasts were fused with PEG 4000. The high-yeilding fusants which have stronger inhibitory activity to Verticillium dahliae V41 and Streptomyces scabies H2 than Streptomyces roseoflavus Men-myco-93-63 were screened. The stability of fusants was detected by subcultured and the characteristics of culture and morphology was observed. The genetic relationship to parents was verified by genome digestion and electrophoresis of soluble proteins. The yield of antibiotics crude produced by high-yeilding strains was obtained by shaking fermentation and the increase rate was got. The shaking fermentation experiment of Fusant RH19 was carried out using the medium of parent, and the antibiotics crude of Fusant RH19 was analyzed with TLC and HPLC methods. We got 14 fusants which have inhibition effect to Verticillium dahliae V41 and Streptomyces scabies H2 through protoplast fusion between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24. The inhibition effect of Fusant RH19 was significantly higher than Streptomyces roseoflavus Men-myco-93-63. The high-yielding fusant RH19 produced yellowish pigments in solid R2YE medium and liquid oats medium, and the characteristics of culture and morphology were different from parental strains. After subcultured 10 times, the fusants have stable heredity. The digestion of genome showed that the restriction fragments differ from that of parents. SDS-PAGE analysis showed that the soluble proteins of Fusant RH19 were different from that of parents, and noval bands were produced. The flasking fermentation experiment result showed that the yeild of antibiotics crude of Fusant RH19 was 0.18 g/L, and that of Streptomyces roseoflavus Men-myco-93-63 was 0.07 g/L, with 159% increase rate. TLC and HPLC results showed that Fusant RH19 produced new secondary metabolites that parent strain could not. This study confirmed that genome combination occured between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24. The research results would provide new resources for prevention of crop diseases using actinomycosis, and it has a broad prospect of application and important application value.This study explored the feasibility of electroporation between Streptomyces roseoflavus Men-myco-93-63 and Streptomyces lividans TK24, the results showed that the method of protoplast fusion using electroporation could not obtain recombinants which has superior character, the condition of electroporation was to be groped.