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 pNSL

摘要:

摘要：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117252 bp的序列，包括在红球菌中保守的1282 bp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767 bp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。

Abstract:

Abstract: 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 4.1040. [Conclusion] Cloning and sequencing of Rhodococcus linear plasmid pNSL1, and identification of its replication origin.