| Spinosad,a secondary metabolite from the fermentation of actinomyces Saccharopolyspora spinosa,is a new type of biological insecticide.With the characteristics of safe and efficient,it has been widely used in many countries.In this study,the stable strain B7 was obtained by the separation and purification of the spinosad-production strains B preservated in laboratory,and the spinosad production was195.43μg/m L（40 m L/250 m L flask）.The strain B7 was selected as an original strains for the mutation breeding.In addition,cerulenin,sodium propionate,monopotassium phosphate and2-deoxidation-D-glucose were used as screening reagents to screen the mutant strains of different resistance directionally and improve the efficiency of breeding.Through the resistance tests of the screening reagents,the screening concentration of cerulenin was determined to be 30μg/m L,the screening concentration of sodium propionate was 4 mg/m L,the screening concentration of monopotassium phosphate was 30 mg/m L and the screening concentration of 32 mg/m L to 2-deoxidation-D-glucose.Starting with the strain B7,four rounds of the atmospheric and room temperature plasma（ARTP）mutagenesis were carried out,and the working gas of mutagenesis was99.99%high purity helium（He）.According to the relationship of mutation dose between with mortality and the relevance between mutation dose and positive mutation rate,the best mutagenic doses were determined to be 50-70 s,and sodium propionate was confirmed to be the most efficient screening reagents.Through four rounds of mutagenesis screening,the stable strain 4-50-6 was obtained and the spinosad production achieved 375.85μg/m L（40m L/250 m L flask）,which was increased by 92.32%to the original departure strain.The composite mutagenesis of plasma and ultraviolet was conducted to the strain 4-50-6 to obtain high-yield strains by the multifunctional plasma mutagenesis system（MPMS）,and the working gas of mutagenesis was 99.99%high purity nitrogen（N2）.The study confirmed that the exposure dose of ultraviolet was 30s and the doses of plasma were 10-40s,and the stable strain 5-35-19 was obtained by the screening of sodium propionate.The spinosad production of strain 5-35-19 was 441.86μg/m L（40 m L/250 m L flask）,which was increased by 126.10%compared with the original departure strains.The study of the relationship between colony morphology of mutant strains and the production confirmed that there is a certain correlation between them.It will be more efficient to screen the improved strains by using the corresponding relationship. |
