肉色吸水链霉菌SH121接合转移系统的建立
State Key Laboratory of Agricultural Microbiology,Huazhong Agricuitural University,Wuhan 430070,China

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MgCl2浓度对链霉菌SH121接合转移效率的影响，结果显示：在50℃热激10 min，37℃预萌发1 h，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40 mmol/L MgCl2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,pregermination time,donor hosts,ratio of donor and recipient and MgCl2 concentration in medium on conjugation efficiency were studied by single factor experiments.Under heat treatment at 50℃ for 10 min,pregermination at 37℃ for 1 h,using Escherichia coli DCDA/pUZ8002 as donor host,with the ratio of donor to recipient being 100 to 1,supplementing MISP4 medium with 40 mmol/L MgCl2,a highly efficient intergeneric conjugation method for SH121 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 intoSH121 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 SH121.