高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

从猪粪堆肥中分离到一株编号为X3-3的可以在50℃高温生长的链霉菌菌株,该菌株含有一个约7kb的环型质粒pTSC2。【目的】克隆、测序和分析pTSC2,以及鉴定质粒的复制方式。【方法】利用分段克隆和引物延伸获得pTSC2的全序列,利用多序列比对寻找复制元件rep、dso和sso,利用中性转移和Southern杂交检测复制中间体。【结果】克隆和测序获得了全长为7516bp的pTSC2序列,预测编码8种蛋白,其中4种蛋白与链霉菌滚环复制的质粒pIJ101中负责复制和接合转移的蛋白非常相似。pTSC2的复制元件rep、dso和sso也与pIJ101的相似。克隆、转化变铅青链霉菌ZX7以及高温链霉菌2C证明了rep和dso为复制所必需元件。Southern杂交检测到pTSC2复制过程中积累了大量的单链DNA。【结论】高温链霉菌质粒pTSC2以滚环方式进行复制。这是首次在高温链霉菌中克隆和测序质粒,以及鉴定其复制方式。

Sequencing and analysis of thermophylic Streptomyces plasmid pTSC2 and characterization of its rolling-circle replication

Objective] A Streptomyces strain X3 -3, growing up to 50 ℃, was isolated from the composts prepared from swine manure and straw. This strain harbored a -7-kb plasmid pTSC2. Objective ] Cloning, sequencing and analyzing pTSC2, and identifying its replication mode as well. Methods ] The complete nucleotide sequence of pTSC2 was obtained by sub-cloning and primer-walking ; the replication gene (rep) , double-strand origin (dso) and single-strand origin (sso) were identified by multiple sequence alignments; and the replication intermediate was detected by Southern hybridization after neutral transfer. Results] The complete nucleotide sequence of pTSC2 consisted of 7516-bp in length, encoded eight proteins, four of which resembled the replication and conjugation proteins of Streptomyces plasmid pIJ101 which replicates in a rolling-circle mode. The replication elements, rep, dso and sso, were also similar with pIJ101. The rep and dso was proved as essential components for plasmid replication by transformation of both S. lividans ZX7 and thermophilic Streptomyces spp. 2C, while sso was dispensable for replication. Southern hybridization detected a large amount of single-stranded DNA accumulated during replication. Conclusion ] We identified that the thermophilic Streptomyces plasimd pTSC2 replicated in a rolling-circle mode. It was first reported that the plasmid from thermophilic Streptomyces was cloned and sequenced, as well as the replication of which was characterized