Streptomyces lavendulae X33遗传转化体系的建立及其内源性启动子活性的比较

Establishment of Genetic Transformation System for Streptomyces lavendulae X33 and Activity Comparison of its Endogenous Promoters

【目的】通过对生防菌淡紫灰链霉菌X33(Streptomyces lavendulae X33)建立稳定高效的遗传转化体系、评估启动子permE与内源性启动子启动活性,为菌株X33活性产物的生物合成机制研究、构建高产基因工程菌奠定基础。【方法】以携带整合型质粒pIB139的大肠杆菌(Escherichia coli)ET12567(pUZ8002)为供体菌、菌株X33为受体菌进行接合转移;对影响接合转移效率的关键因素(接合转移培养基、热激温度、预萌发时间、供受体比例及抗生素覆盖时间等)进行优化;以质粒pIB139为骨架、β-葡萄糖苷酶(GUS)为报告基因构建启动子活性检测载体,分析4个内源性启动子及permE的启动活性。【结果】通过接合转移方法成功建立了菌株X33的遗传转化体系,其最佳接合转移条件为:孢子于55℃热激10 min,预萌发1 h,供、受体比例为10∶1,以高氏一号为接合转移培养基,混合培养12 h后覆盖抗生素,接合转移效率可达5.8×10^(-5);启动子活性分析表明:链霉菌常用启动子permE的启动活性远高于4个内源性启动子P116、P4565、P5092、P5500。【结论】建立及优化了菌株X33的遗传转化体系,确定了启动子permE在菌株X33中的启动活性。

[Objective]This study aims to provide better foundations for the research of biosynthesis mechanism of bioactive product in Streptomyces lavendulae X33 and the construction of high-yield strains by establishing genetic transformation system for Streptomyces lavendulae X33 and evaluating its endogenous promoters activity.[Methods]With the two strains of Escherichia coli ET12567(pUZ8002),carrying the integrated plasmid pIB139 as the donor and strain X33 as the recipient,the conjugation transfer was performed.The key factors affecting the efficiency of conjugation transfer,including conjugation transfer medium,heat shock temperature,pre-germination time,donor & recipient ratio and antibiotic coverage time,were optimized.Using plasmid pIB139 as the skeleton and β-glucosidase(GUS)as the reporter gene,the promoter activity of four endogenous promoters and permE was analyzed.[Results]The genetic transformation system of strain X33 was successfully established with conjugation transfer method.The optimal conjugation transformation with a conjugation transformation efficiency of 5.8×10^(-5) was realized under the following conditions:spores incubation at 55 ℃ for 10 min,pre-germination for 1 h,10∶1 for donor to recipient ratio,Gauze’s medium No.1 as conjugation transfer medium,and covering antibiotics after mixed culture for 12 h.The analysis of promoter activity showed that the activity of permE,commonly used as Streptomyces promoter,was much higher than that of the four endogenous promoters P116,P4565,P5092 and P5500.[Conclusion]A genetic transformation system of strain X33 was established and optimized,and the activity of the promoter permE was determined in strain X33.