羽毛降解杆状链霉菌S-28遗传转化系统的建立与优化

Establishment and optimization of genetic transformation system for feather-degrading strain Streptomyces bacillaris S-28

【目的】建立羽毛降解杆状链霉菌(Streptomyces bacillaris)S-28的遗传转化系统,并对其进行优化,为该菌株的遗传改造奠定基础。【方法】以大肠杆菌-链霉菌穿梭整合型质粒pSET152为出发质粒,大肠杆菌(Escherichia coli)S17-1/pSET152为供体,羽毛降解杆状链霉菌S-28孢子为受体进行接合转移试验,并对影响接合效率的培养基种类、MgCl_2浓度、热激温度、预萌发时间、供受体比例和接合转移时间等要素进行优化。【结果】确定筛选羽毛降解杆状链霉菌S-28菌株接合转化子的抗生素为安普霉素≥10μg/mL或卡那霉素≥20μg/mL;成功将pSET152转入到杆状链霉菌S-28中。优化的S-28菌株接合转移条件为:以MS为接合转移最适培养基,MgCl_2浓度为10mmol/L,孢子萌发条件为40℃热激10min且不进行预萌发处理,供受体比例为10∶1,接合转移时间18~20h,此条件下接合转化效率最高达到10-4。【结论】建立并优化了羽毛降解杆状链霉菌S-28的遗传转化系统,接合转化效率最高可达到10^(-4)。

【Objective】The genetic transformation system of feather-degrading strain Streptomyces bacillaris S-28 was established and optimized to provide basis for genetic modification of the strain.【Method】In this article,the shuttle integrated plasmid pSET152 was used as starting plasmid fromEscherichia coli into Streptomyces,E.coli S17-1/pSET152 was used as the donor and feather-degrading strain Streptomyces bacillaris S-28 spore was used as the receptor.Then,the conjugal transfer of pSET152 fromE.coli S17-1 into S-28 was conducted.Factors including culture medium,concentration of MgCl2,heat temperature,pre-germination time,donor-to-recipient ratio and conjugation time were optimized.【Result】The selected antibiotic was Apramycin≥10μg/mL or Kanamycin≥20μg/mL for screening ex-conjugants of the feather-degrading strain Streptomyces bacillaris S-28 and the plasmid pSET152 was successfully transferred into Streptomyces bacillaris S-28.The optimized conjugation transfer conditions of S-28 were conjugation transfer medium MS,MgCl2 concentration 10 mmol/L,heat shock 40 ℃ for 10 min without pre-germina-tion,donor-to-recipient ratio 10∶1,and coverage time 18-20 h.The best conjugation efficiency was up to10^（-4）.【Conclusion】The conjugation transfer system was established and optimized successfully with the transfer efficiency of 10^（-4） under optimal conditions.