肉色吸水链霉菌SH121接合转移系统的建立

Establishing intergeneric conjugation system in Streptomyces carneohygroscopicus SH121

利用单因素试验探索了热激处理、预萌发时间、供体宿主菌的类型、供受比及MgCl_2浓度对链霉菌SH121接合转移效率的影响,结果显示:在50℃热激10min,37℃预萌发1h,以大肠杆菌DCDA/pUZ8002为供体宿主菌,在供受比为100∶1时,使用添加40mmol/L MgCl_2的培养基M-ISP4,接合转移效率最高可达到每受体1.03×10^(-3)个接合子。利用建立好的接合转移方法,将整合型质粒pSET152和基因敲除质粒pYZ09成功地导入链霉菌SH121,证实该方法可用于后期链霉菌SH121活性天然产物的挖掘及其相关生物合成基因簇的研究工作。

Streptomyces carneohygroscopicus SH-121 isolated during selecting biocontrol agent of citrus diseases exhibits strong inhibition towards Gram positive bacteria and many pathogenic fungi in plant.Establishment of a highly efficient genetic manipulation system is critically required for functionally analysing genes and genetic engineering in Streptomyces.In this study,the effects of heat treatment,pre-germination time,donor hosts,ratio of donor and recipient and MgCl_2 concentration in medium on conjugation efficiency were studied by single factor experiments.Under heat treatment at 50 ℃ for 10 min,pre-germination at 37℃for 1h,using Escherichia coli DCDA/pUZ8002 as donor host,with the ratio of donor to recipient being 100 to 1,supplementing M-ISP4 medium with 40mmol/L MgCl_2,a highly efficient intergeneric conjugation method for SH-121 was developed with highest efficiency of 1.03×10^(-3)conjugants/recipient.The integrative plasmid pSET152 and the plasmid pYZ09 for gene knockout was successfully transferred into SH-121 with the method newly established.It is indicated that this genetic manipulation system could be used to discover bioactive natural products and study their biosynthetic gene clusters in SH-121.