南海红树林来源链霉菌Streptomyces sp.OUC6819遗传操作系统的建立  

Development of the genetic system of mangrove-derived Streptomyces sp.OUC6819

作　　者：刘增智[1] 李通[1] 刘晶[1] 姚婷婷[1] 张慧[1] 夏娟[1] 李花月 车茜[1] 李文利[1] 

机构地区：[1]中国海洋大学教育部海洋药物重点实验室医药学院,山东青岛266003

出　　处：《中国海洋药物》2016年第4期53-59,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金项目(31171201;31570032)资助

摘　　要：目的建立南海红树林来源链霉菌Streptomyces sp.OUC6819菌株的遗传转化系统,通过基因阻断方法来研究次级代谢产物的生物合成途径。方法分别以整合型载体pSET152和用于同源重组的、包含可能参与大环多烯类化合物生物合成的DNA片段的非复制型cosmid质粒,通过大肠杆菌—链霉菌属间接合转移导入Streptomyces sp.OUC6819之中。结果通过在接合转移过程中对菌丝体进行超声破碎,成功获得了正确的接合子。采用PCR方法验证了pSET152的导入和目标基因orf1的阻断,进一步对突变株的发酵产物进行了HPLC分析,结果显示突变株丧失了合成大环多烯类化合物的能力。结论成功建立了基于菌丝体超声破碎的海洋链霉菌遗传转化系统,为对其它基因进行遗传学操作奠定了基础,也为其它不便进行遗传操作的链霉菌提供了参考。Objective To develop the genetic system of mangrove-derived Streptomyces sp.OUC6819 and study secondary metabolites biosynthetic pathway via gene disruption.Methods E.coli-Streptomyces intergeneric conjugation was performed to transfer the integrative vector pSET152 and suicide cosmid,which harbored genes putatively involved in the biosynthesis of macrolide polyene compounds,into Streptomyces sp.OUC6819.Results Exconjugants were successfully obtained efficiently by using ultrasonic fragmented myceliaas conjugation receptors.Exconjugants were verified using PCR methods.The mutant strain was further fermented and analyzed by using HPLC analysis,which revealed that the mutant strain lost the ability to produce macrolide polyene compounds.Conclusion Conjugation-based genetic system of marine-derived Streptomyces sp.OUC6819 was successfully established using ultrasonic fragmented mycelia as acceptors,which laid the foundation for genetic manipulation of this stain,and more importantly,provided important reference for other Streptomyces strains difficult to be handled genetically.

关 键 词：STREPTOMYCES sp.OUC6819 PKSs PCR-targeting 接合转移 基因敲除 

分 类 号：R915[医药卫生—微生物与生化药学]