链霉菌小质粒pDYM4.3k的复制与接合转移

Replication and conjugation of Streptomyces small plasmid pDYM4. 3k

摘要:

摘要:【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。

Abstract:

Abstract:［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．