黄色链霉菌TRM45540遗传转化系统的构建

Construction of the genetic transformation system for Streptomyces luteus TRM45540

黄色链霉菌(Streptomycesluteus)TRM45540是分离自新疆罗布泊土壤的高产放线菌素D的链霉菌新物种,为了深入挖掘其基因功能和代谢活性,需要建立该菌株的遗传转化系统。本研究以整合型载体pSET152和自杀型载体pKC1139为出发质粒,ET12567(pUZ8002)为供体菌,TRM45540为受体菌,通过大肠杆菌-链霉菌属间接合转移方法建立TRM45540遗传转化系统。MS培养基为最适产孢培养基和接合子最适生长培养基,TRM45540菌株对安普霉素(APR)、硫链丝菌素(THIO)和卡那霉素(KAN)敏感;TSB培养基为大肠杆菌-黄色链霉菌属间接合转移最适培养基;最适萌发时间为4 h。结果表明:TRM45540菌株接合转移系统通过MS培养基培养收集孢子,将收集的孢子于TSB培养基中50℃热激10 min,37℃萌发4 h后,与供体菌ET12567(pUZ8002)在MS培养基上进行接合转移,接合转移的抗性筛选标记可以采用APR抗性、THIO抗性和KAN抗性。本研究成功构建TRM45540菌株的遗传转化系统,为后期进行遗传转化研究奠定了基础。

Streptomyces luteus TRM45540 is a new species produces actinomycin D efficiently isolated from Lop Nur soil in Xinji-ang,to further study its gene function and metabolic activity in Streptomyces luteus TRM45540,the genetic transformation system for this strain were constructed.In this research,the integrating vector pSET152 and suicide vector pKC1139 were used as the start-ing plasmid,ET12567(pUZ8002)used as the donor,TRM45540 used as the receptor,respectively.The genetic transformation sys-tem of Streptomyces luteus was established by the method for junction transfer between genus of Escherichia coli-Streptomyces.MS medium was the best sporulation and zygote growth medium,and TRM45540 strain was sensitive to apramycin,thiostrepton and kanamycin.TSB medium was the optimal medium for junction transfer between genus of Escherichia coli-Streptomyces.The optimal germination time was 4 h.The results show that:The optimum spore-producing medium for Streptomyces luteus TRM45540 and Escherichia coli junction transfer was MS medium after spores were heat shocked in TSB medium at 50℃for 10 min and germina-tion at 37℃for 4 h,then conjugated and transferred with the donor ET12567(pUZ8002)on MS medium,and clarified antibiotics apramycin,thiostrepton and kanamycin can be used as screen markers to filtrate the transconjugants of Streptomyces luteus TRM45540.therefore the genetic transformation system of Streptomyces luteus TRM45540 was successfully established,which laid a foundation for the later genetic transformation research.