尤马马杜拉放线菌染色体复制区的克隆及功能分析

Cloning and functional studies of the chromosomal replication origin of Actinomadura yumaensis

摘要:

尤马马杜拉放线菌(Actinomadura yumaensis)NRRL12515产生马杜拉霉素,用于防治禽类球虫病。试验以放线菌dnaA与dnaN基因保守区设计的简并引物进行PCR扩增,获得了包含尤马马杜拉放线菌的染色体复制区oriC的片段,并进行了序列分析和复制功能的研究。尤马马杜拉放线菌染色体的oriC全长为919碱基对,含有14个DnaA盒子和2个AT富含区,DnaA盒子的保守序列是(T/C)(T/C)GTCC(A/C)CA,与已发表的3个属的放线菌染色体oriC的序列特征不同。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制并以低拷贝方式遗传,表明这是一段有复制功能的序列。比较来自放线菌4个属的oriC,发现以oriC序列和以16S rRNA基因序列构建的进化树十分相似,表明oriC序列也可以体现放线菌物种之间的关系。

Abstract:

The chromosomal replication origins (oriC) have been investigated in Gram-positive eubacteria actinomycetes，including Streptomyces，Mycobacterium and Amycolatopsis，and reveal various DnaA-boxes and AT-rich sequences between the conserved dnaA and dnaN genes. Actinomadura yumaensis NRRL12515 is a producer of anthelmintic polyether maduramicin. In this paper，cloning，sequencing and functional studies of its oriC have been carried out. A pair of oligo-nucleotide primers，based on the conserved sequences of dnaA and dnaN，was used to PCR amplification. A～1.3kb DNA band was detected on agarose gel. Subsequently cloning in an E. coli plasmid pBluescriptⅡ SK(+) and sequencing showed 1265bp，which contained 919bp between dnaA and dnaN genes. 14 DnaA-boxes with conserved 9bp sequence (T/C)(T/C)GTCC(A/C)CA and two 13bp AT-rich regions (GAAAAATCCCAAG，AAGAAAAAACTCA)，were found on the sequence，indicating the oriC of A. yumaensis NRRL12515. Phylogenetic trees based on the sequences of oriC and of 16S rRNA genes of the four actinomycetes species show a similar pattern，suggesting that oriC sequences also reflected well the relationship between actinomycetes species. An E. coli plasmid pOR1，containing the oriC，actinomycetes selection markers tsr and melC，was introduced into Streptomyces coelicolor M145 by conjugal transfer. Transformants were obtained，and plasmids DNA were isolated and detected as low copy number，suggesting a functional mini-chromosome in Streptomyces.