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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 establish a genetic transformation system of Streptomyces sp. OUC6819 strain from the mangroves in the South China Sea, and to study the biosynthetic pathway of secondary metabolites through gene blockade. Methods Streptomyces sp was transferred into Escherichia coli-Streptomyces sp. By non-replicating cosmid plasmid containing homologous recombination and DNA fragments which may participate in the biosynthesis of macrocyclic polyene respectively. OUC6819 among them. Results The correct conjugate was successfully obtained by sonicating the mycelium during conjugation transfer. PCR was used to verify the introduction of pSET152 and blockade of the target gene orf1. Further analysis of the fermentation products of the mutant strains by HPLC showed that the mutant strains lost the ability to synthesize macrocyclic polyene compounds. Conclusion The genetic transformation system of S. marinus based on mycelial ultrasonic disruption has been successfully established, which laid the foundation for the genetic manipulation of other genes and provided references for other streptomyces which are inconvenient for genetic manipulation.