Journal
MOLECULAR PLANT PATHOLOGY
Volume 22, Issue 7, Pages 843-857Publisher
WILEY
DOI: 10.1111/mpp.13058
Keywords
Burkholderia pyrrocinia; freeze‐ thawing transformation; GC‐ rich gene; homologous recombination; knockout
Categories
Funding
- Jiangsu Agricultural Science and Technology Independent Innovation Fund [CX16-1005]
- Chinese State Forestry Administration Special Research Program for Forestry Sectors Beneficial to the Public [201304404]
- Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)
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A novel genetic transformation system was developed to knock out a highly GC-rich gene from Burkholderia pyrrocinia JK-SH007. This system included a reliable selectable marker and an efficient transformation method, ultimately leading to the creation of a mutant strain with significantly reduced ability to degrade carboxymethyl cellulose. Overall, this system has the potential to further research on traits of Burkholderia pyrrocinia.
Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC-rich gene (74.71% GC) from Burkholderia pyrrocinia JK-SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/mu g, pHKT2) that was developed via freeze-thawing. The knockout recombineering of B. pyrrocinia JK-SH007 was achieved through a suicide plasmid with a three-fragment mutagenesis construct. The three-fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC-rich upstream and downstream fragments from B. pyrrocinia JK-SH007. The mutant strain (Delta BpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits.
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