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/μg, 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 (ΔBpEG), 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.

中文翻译：

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通过重组工程与冻融转化敲除吡咯氏伯克霍尔德菌中高 GC 含量的基因

遗传转化是一种有价值且必不可少的方法，它提供了对微生物基因功能的有力见解，并有助于工程细菌的构建。在这里，我们开发了一种新的遗传转化系统，可以轻松地从Burkholderia pyrrocinia JK-SH007（​​一种杨树溃疡病的生物防治菌株）中敲除高 GC 含量的基因（74.71% GC）。该系统揭示了可靠的选择标记（甲氧苄啶抗性基因，Tmp）和通过冻融开发的简化、高效的转化方法（6,363.64 CFU/μg，pHKT2）。B. pyrrocinia的敲除重组工程JK-SH007 是通过具有三片段诱变构建体的自杀质粒实现的。用于诱变的三片段盒是通过重叠延伸和着陆 PCR 产生的，由Tmp组成，两侧是来自B.pyrrocinia JK-SH007的富含 GC 的上游和下游片段。经 PCR 验证的突变菌株 (Δ BpEG ) 在 40 天内失去了 93.3% 的降解羧甲基纤维素的能力。总体而言，该系统可能有助于未来对B. pyrrocinia性状的研究。