| 摘要: |
| 【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、MgCl2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≥10 μg/mL或卡那霉素≥20 μg/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，MgCl2浓度为10 mmol/L，孢子萌发条件为40 ℃热激10 min且不进行预萌发处理，供受体比例为10∶1，接合转移时间18~20 h，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。 |
| 关键词: 杆状链霉菌S-28 羽毛降解 遗传转化系统 接合转移 |
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| 基金项目:中央高校基本科研业务费专项(Z109021426,Z109021432)；陕西省农业科技创新与攻关项目（2015NY066） |
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| Establishment and optimization of genetic transformation system for feather-degrading strain Streptomyces bacillaris S-28 |
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ZHANG Jinlong,MA Yiming,SHU Weixue,et al
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| Abstract: |
| 【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 from Escherichia 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 from E.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≥10 μg/mL or Kanamycin≥20 μg/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-germination,donor-to-recipient ratio 10∶1,and coverage time 18-20 h.The best conjugation efficiency was up to 10-4.【Conclusion】The conjugation transfer system was established and optimized successfully with the transfer efficiency of 10-4 under optimal conditions. |
| Key words: Streptomyces bacillaris S-28 feather-degrading genetic transformation system conjugal transfer |
