红球菌线型质粒pNSL1的克隆、测序和复制区的鉴定

Cloning,sequencing and identification of replication origin of Rhodococcus linear plasmid pNSL1

从红球菌NS1中检测到两个线型质粒pNSL1和pNSL2。【目的】克隆、测序和分析pNSL1,并鉴定质粒的复制区。【方法】利用脉冲电泳方法从凝胶中回收大量的质粒DNA,进行鸟枪法克隆、测序和拼接,通过生物信息学分析和实验证明质粒的自主复制区。【结果】克隆、测序和拼接获得pNSL1全长为117252bp的序列,包括在红球菌中保守的1282bp端粒的序列。序列预测含有103个蛋白编码区,包括质粒的复制、分配、转移等功能基因。将pNSL1中一个与分枝杆菌质粒的复制基因同源的pNSL1.038及其上游的767bp非编码序列克隆到大肠杆菌质粒,电击转化珊瑚诺卡氏菌4.1037,获得了抗性转化子。【结论】克隆、测序了全长的线型质粒pNSL1,鉴定了质粒的复制区。

Two linear plasmids,pNSL1 and pNSL1,were detected from Rhodocuccus sp.NS1.[Objective]Cloning,sequencing and identification of replication origin of the Rhodococcus linear plasmid pNSL1.[Methods]Large amount of linear plasmid DNA was recovered from pulsed-field gels for shotgun-cloning and sequencing,and identification of its replication locus.[Results]The complete nucleotide sequence of pNSL1 consisted of 117252 bp,including the conserved 1282-bp telomere sequences among Rhodococcus linear plasmids.pNSL1 encoded 103 open reading frames,including functions of replication,maintenance and transfer etc.A locus,pNSL1.038 and upstream 767-bp non-coding sequence,was identified for autonomous replication by cloning in an E.coli vector and introduced by electroporation into Nocardia coralline 4.1037.[Conclusion]Cloning and sequencing of Rhodococcus linear plasmid pNSL1,and identification of its replication origin.