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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis
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Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis

机译：从Couchioplanes Caeruleus分离新的质粒和两个质粒载体的基因表达施工issouriensis

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摘要

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis. (C) 2014 Elsevier Inc. All rights reserved.
机译：迄今为止，还没有为罕见的放线菌密苏里放线菌开发质粒载体。此外，在放线菌属中还没有发现小的环状质粒。在这里，一个新的质粒，命名为pCAZ1，是从库奇奥普兰蓝亚种中分离出来的。azureus通过在放线菌（57株）和古氏菌（2株）中筛选小的环状质粒。核苷酸测序显示pCAZ1是一个5845bp的环状分子，G+C含量为67.5%。pCAZ1的拷贝数估计为每个染色体30个。pCAZ1包含七个假定的开放阅读框，其中一个编码一个包含三个基序的蛋白质，这些基序保存在质粒编码的复制蛋白质中，这些复制蛋白质参与复制的滚动循环机制。通过检测C.caeruleus中的单链DNA中间产物，证实pCAZ1通过这种机制复制。将来自pBluescript SK（+）的ColE1序列和来自pIJ773的安普霉素抗性基因aac（3）IV的oriT序列一起插入pCAZ1，以构建大肠杆菌密苏里梭菌穿梭载体pCAM1和pCAM2，其中外源DNA片段以相反方向插入pCAZ1。pCAM1和pCAM2通过大肠杆菌介导的接合转移系统成功转移到密苏里A.missouriensis。据估计，密苏里A.的pCAM1和pCAM2的拷贝数分别为每个染色体1和4个。因此，这些载体可以作为密苏里A.同源和异源基因表达研究的有效遗传工具。（C） 2014爱思唯尔公司版权所有。

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