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以经理化诱变选育的多杀菌素高产菌株刺糖多孢菌(Saccharopolyspora spinosa)CB11为受体菌,将具有安普霉素(apramycin)抗性标记的整合型载体pSET152作为质粒供体,研究了制备刺糖多孢菌感受态细胞时菌体的生长阶段、质粒DNA浓度、电场强度等因素对电转化效率的影响,结果表明,在菌体培养48h至对数中期制备感受态,电场强度为12kV/cm,最小DNA浓度为0.1μg时可获得最高的转化效率,转化子的安普霉素抗性基因的PCR扩增及抗性稳定性实验从分子水平及细胞水平验证了pSET152质粒能整合在刺糖多孢菌的基因组中并能稳定遗传,所有结果表明所建立的刺糖多孢菌电转化体系是行之有效的。
The shuttle plasmid pSET152, which has apramycin resistance marker, was used as the recipient strain of the spinosad strain CB11, a spinosad-producing strain of spinosad, The effects of cell growth stage, plasmid DNA concentration, electric field intensity and other factors on the efficiency of electroporation were studied. The results showed that during the period from 48h culture to mid-logarithmic phase preparation, the electric field The highest transformation efficiency was obtained when the concentration of DNA was 0.1μg at the intensity of 12kV / cm. PCR amplification and resistance stability test of the apomycin resistance gene of the transformants verified the pSET152 plasmid at the molecular and cellular levels Can be integrated into the genome of S. spinosa and can be stably inherited. All the results show that the established S. spinosa electroporation system is effective.