链霉菌小质粒pDYM4.3k的复制与接合转移

【目的】链霉菌(Streptomyces)X335是从西藏高原活拉山口分离到的,其中含有一个大小为4.3 kb的环型质粒pDYM4.3k。克隆、测序和分析pDYM4.3k,以及鉴定复制和接合转移的基因。【方法】通过克隆和引物延伸获得pDYM4.3k的全序列,利用比对分析推测基因的功能,通过Southern杂交检测复制中间体,利用平板杂交实验证明接合转移功能。【结果】克隆和测序获得了全长为4346 bp的pDYM4.3k序列,预测仅有3个基因,其中1个基因与链霉菌主要接合转移基因同源,另外2个为功能未知。鉴定新的基因orf1及其上游的约300 bp构成了质粒的基本复制区域。检测到质粒存在单链的复制中间体,表明它以滚环方式进行复制。实验证明pDYM4.3k在变铅青链霉菌(Streptomyces lividans)中具有接合转移功能。【结论】质粒pDYM4.3k可以滚环方式进行复制和在链霉菌之间进行接合转移。这是目前报道的最小的、具有游离复制和接合转移功能的链霉菌质粒。

Replication and conjugation of Streptomyces small plasmid pDYM4. 3k

Objective] Streptomyces sp.X335,harboring a 4.3-kb plasmid pDYM4.3k,was isolated from Tibet sample.Cloning,sequencing,analyzing and functional investigations of pDYM4.3k.Methods] Prime walking to obtain plasmid sequence,BLAST to predict gene function,Southern hybridization to determine replication intermediates,and mating to test conjugal function of plasmid.Results] The complete nucleotide sequence of pDYM4.3k consisted of 4346 bp,encoding 3 genes of which one resembled Streptomyces major conjugative gene tra and other two were with unknown functions.Experiments demonstrated that a new gene of pDYM4.3k and its upstream c.300 bp were required for plasmid replication.Single-stranded pDYM4.3k as a replication intermediate was detected,indicating it rolling-cirle replication mode.pDYM4.3k could conjugal transfer among Streptomyces lividans.Conclusion] Streptomyces small plasmid pDYM4.3k was able to autonomous replication and conjugal transferring.