藤黄生孢链霉菌NRRL2401遗传操作系统和基因文库的构建

Genetic manipulation system and genomic library of Streptomyces luteosporeus NRRL 2401

摘要:

[目的] 建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。[方法] 利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^TM载体,大肠杆菌EPl300TM-T1R为宿主菌构建Fosmid文库。随后,设计引物,利用“板池-行池-单克隆”的三级PCR方法对文库进行快速筛选。[结果] pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。[结论] 成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。

关键词:藤黄生孢链霉菌;接合转移;基因文库;PCR筛选

Abstract:

[Objective] To clone the biosynthetic gene clusters for secondary metabolites, we developed the genetic modification system and constructed a genomic library of Streptomyces luteosporeus NRRL 2401. [Methods] The genetic modification system was developed by using conjugal transfer vectors pSET152, pPM927 and pJTU1278 which were transferred from Escherichia coli ET12567/pUZ8002 to S. luteosporeus. The genomic library of S. luteosporeus NRRL 2401 was constructed by the fosmid vector pCClFOS^TM, with E. coli EPl300TM-T1R as the host strain. A PCR-based method was then developed for screening the biosynthetic gene clusters of secondary metabolites in the constructed genomic library. [Results] Vectors pSET152, pPM927 and pJTU1278 were successfully transferred into S. luteosporeus for genetic modification, with pSET152 presenting the highest transformation efficiency. The constructed genomic library of S. luteosporeus NRRL 2401 contained 2880 clones with an average -35 kb inserted DNA fragment in each clone, indicating the 99.99% coverage of the genome in the library. In this genomic library, we detected 9 clones containing possible indolmycin biosynthesis genes by the PCR-based screening method. [Conclusion] A stable, efficient genetic modification system and high-quality genomic library could be used for discovery of the biosynthetic gene clusters for secondary metabolites in S. luteosporeus NRRL 2401.

Key words:Streptomyces luteosporeus;conjugal transfer;genomic library;PCR-based screening