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黄单胞菌的高压电穿孔转化和黄单胞菌克隆载体的构建。

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors.

机构信息

Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Ghent, Belgium.

出版信息

Appl Environ Microbiol. 1990 Sep;56(9):2818-25. doi: 10.1128/aem.56.9.2818-2825.1990.

DOI:10.1128/aem.56.9.2818-2825.1990
PMID:16348290
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC184849/
Abstract

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10/mug of DNA to 10/mug of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.

摘要

植物病原菌罗尔斯通氏菌毒力决定因子的分析一直受到缺乏外源 DNA 导入系统的阻碍。我们通过在存在质粒 DNA 的情况下对完整细菌细胞进行高压电穿孔，研究了罗尔斯通氏菌遗传转化的可能性。在罗尔斯通氏菌 D188 中的电转化导致转化频率范围从每微克 DNA 10 到每微克 DNA 10，具体取决于 DNA 浓度。介绍了不同电参数和电穿孔介质组成对转化效率的影响。通过这种转化方法，基于来自菌株 NCPPB 1675 的本土 160 千碱基（氯霉素和镉抗性编码）质粒 pRF2 的罗尔斯通氏菌克隆载体（pRF28）得以开发。pRF28 上的复制起点和氯霉素抗性基因被用于构建能够在罗尔斯通氏菌和大肠杆菌中复制的克隆载体。所提出的电穿孔方法效率足够高，能够通过同源或非同源重组检测到复制缺陷型 pRF28 衍生物在罗尔斯通氏菌 D188 基因组中的罕见整合。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d40/184849/1efbed845e24/aem00090-0244-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d40/184849/b2dd28540f49/aem00090-0243-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d40/184849/1efbed845e24/aem00090-0244-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d40/184849/b2dd28540f49/aem00090-0243-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0d40/184849/1efbed845e24/aem00090-0244-a.jpg

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