Detection and characterization of a theta-replicating plasmid pLP60 from Lactobacillus plantarum PC518 by inverse PCR
| Autor: | Qu Pan, Xin Du, Fang Yao, Xu Xiaoyu, Cao Kang |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Molecular biology Theta replication Inverse PCR Microbial genomics Biology Plasmid Microbiology Microbial biotechnology DNA sequencing Article 03 medical and health sciences Complete sequence 0302 clinical medicine Tandem repeat Bacterial genetics Genetics ORFS lcsh:Social sciences (General) lcsh:Science (General) Multidisciplinary Long tandem repeats Copy number Inverse polymerase chain reaction Open reading frame 030104 developmental biology Microsatellite lcsh:H1-99 030217 neurology & neurosurgery Lactobacillus plantarum lcsh:Q1-390 |
| Zdroj: | Heliyon, Vol 5, Iss 8, Pp e02164-(2019) Heliyon |
| ISSN: | 2405-8440 |
| Popis: | Plasmid DNA of Lactobacillus plantarum PC518 was isolated by an improved method which contained a washing step for removing lysozyme. Three plasmid DNA libraries were constructed. A pair of outward primers was designed at both ends of the novel plasmid fragment obtained from plasmid DNA libraries, and the remainder of the circle plasmid was amplified by inverse PCR (iPCR). The whole sequence of plasmid was analyzed by the basic local alignment search tool, Tandem Repeats Finder, DNAMAN V6.0, DNASTAR and MEGA X software. The copy number was measured using quantitative real-time PCR. Plasmid extract showed 7 bands on agarose gel, indicating that L. plantarum PC518 contains multiple plasmids. The complete sequence of plasmid pLP60 was obtained by plasmid DNA libraries and iPCR. pLP60 is 6006 bp in length with a G + C content of 41.19 %, which encodes 8 open reading frames (ORFs). The ori site like theta-type could be located upstream of repB, which contains a short tandem repeats (sTR) and a long tandem repeats (lTR). RepB of pLP60 only had low similarity with Rep protein of known theta-type plasmids, but phylogenetic tree analysis showed that plasmids whose Rep proteins are similar to pLP60 have lTR at ori, and the conservativeness of lTR is consistent with similarity of Rep proteins, suggesting that RepB of pLP60 is a theta-replicating protein. So pLP60 was classified as class A of theta replication. The copy number of pLP60 was measured as 5 copies per cell by qPCR. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|