游动双孢菌线型质粒pPR2的全序列测定及分析

Complete nucleotide sequence and analysis of Planobispora linear plasmid pPR2

摘要:

【目的】获得游动双孢菌线型质粒pPR2的全序列，并揭示新型的端粒复制蛋白和可能的中间复制位点。【方法】用分段克隆的方法和序列拼接获得pPR2的全序列，利用软件分析端粒DNA的二级结构和可能的端粒复制蛋白，利用链霉菌原生质体转化的方法检测可能的中间复制的位点。【结果】pPR2全长为15520 bp，（G+C）含量为68.1%。其端粒末端反向重复序列的长度为329 bp，不能像多数链霉菌的线型质粒那样能形成保守的“折返”的二级结构。pPR2虽然没有参与链霉菌端粒复制的保守的tap/tpg基因，但是pPR2.3c基因编码了一个双结构域蛋白，分别同链霉菌的端粒复制相关蛋白Tap和嗜血杆菌的解旋酶具有相似性。pPR2缺少典型的链霉菌重复序列-复制基因（iteron-rep）区段，将几乎覆盖全长pPR2的两段DNA进行克隆后，不能转化变铅青链霉菌。此外，pPR2基因还编码可能参与线型DNA复制的调控的单链结合蛋白（SSB）和与放线菌质粒接合转移相关的主要蛋白(Tra)。【结论】pPR2是链霉菌之外的放线菌中最小的线型质粒，其序列在游动双孢菌属的线型质粒中是首次报道。pPR2可能具有新型的端粒复制的机制，其中pPR2.2c和pPR2.3c编码可能的端粒复制蛋白。

Abstract:

[Objective] To obtain the complete nucleotide sequence of Planobispora linear plasmid pPR2, and reveal the novel telomere replication protein and possible replication loci from the centrally located origin. [Methods] We cloned the EcoRI DNA fragments and assembled them to get the whole sequence of pPR2. We analyzed the secondary structure of the telomere DNA and the putative telomere replication protein using software, and detected the possible replication loci by Streptomyces protoplast transformation. [Results] The complete nucleotide sequence of pPR2 consists of 15520 bp in length, with a 68.1% (G+C) content. The 329 bp terminal inverted repeat sequence can’t form the conserved fold-back secondary structure as that of many Streptomyces linear replicons. Lacking of typical Streptomyces tap/tpg locus for te-lomere replication, pPR2.3c encodes a protein with two domains resembling the telomere associated protein of Strepto-myces and helicase of Haemophilus respectively. No typical Streptomyces iteron-rep locus for replication from the cen-trally located origin, two DNA fragments containing almost all pPR2 were cloned and introduced by transformation into S. lividans ZX7, but no transformants were obtained. pPR2 encodes single-strand binding protein which may regulate repli-cation of linear DNA and Tra protein for conjugation transfer in actinomycetes. [Conclusion] pPR2 is the smallest ac-tinomycete linear plasmid beyond Streptomyces. This is the first time to report the complete nucleotide sequence of linear plasmid in the genus Planobispora. pPR2 may have novel mechanism for telomere replication, and pPR2.2c and pPR2.3c encode the possible telomere replication proteins.