反向筛选标记基因upp在杀真菌链霉菌遗传改造中的应用

Application of Counterseletable Gene upp in Genetic Manipulation of Streptomyces fungicidicus

为进一步简化放线菌的遗传操作流程,缩短重组菌株的筛选周期,在对杀真菌链霉菌(Streptomyces fungicidicus ATCC 21013)进行遗传操作的过程中引入了一个反向筛选标记基因——尿嘧啶磷酸核糖转移酶基因(upp)。并通过原始菌株中upp基因的敲除,以及带有upp基因的自杀型基因敲除载体的构建,开发了一套完整的针对杀真菌链霉菌的无痕敲除系统。通过载体的整合、二次交换及反向筛选实现了杀真菌链霉菌基因组中StrR基因的快速无痕敲除。upp反向筛选标记基因的引入使得链霉菌重组菌株的平均筛选周期缩短了2周左右,并进一步减少了假阳性重组菌株出现的概率,可实现放线菌中目的基因的连续无痕敲除,因此值得进一步推广和应用。

In order to further simplify the genetic operation process of actinomycetes and shorten the screening cycle of the recombinant strains,a counter selectable marker gene——uracil phosphoribosyl transferase gene(upp)was introduced into the genetic operation of Streptomyces fungicidicus ATCC 21013.By deletion of the upp gene in the wild type strain and construction of a suicide vector that carries the upp gene expression cassette,a markerless inframe deletion system was developed,and the StrR gene in S.fungicidicus genome was successfully knocked out after vector integration,plasmid excision and counterselection.The introduction of upp gene shortened the average screening cycle of Streptomyces recombinant strains by about 2 weeks,and further reduced the probability of false positive recombinant strains.The simplicity of this system should make it adaptable for continuous markerless gene deletion in other actinomycetes,so it is worth further promotion and application.