通过在 37°C 和 30°C 温度下交替培养转化株,开发出一种无标记的寄生褐藻菌株遗传操作方法。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY BMC Biotechnology Pub Date : 2024-09-03 DOI:10.1186/s12896-024-00887-w
Jing Xiao, Yuxin Wang, Dongfang Wu, Yuping Song, Xuwang Cai, Huanchun Chen, Hongbo Zhou, Xiaojuan Xu
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引用次数: 0

摘要

背景:寄生格氏菌(Glaesserella parasuis,G. parasuis)是格莱塞氏病的病原体,它给养猪业造成了巨大的经济损失。然而,由于缺乏简单高效的无标记基因敲除系统,对寄生璃泽氏菌发病机制的研究一直受到阻碍:结果:本研究利用对温度敏感的载体为寄生虫开发了一种无标记基因剔除系统。通过交替在 30°C 和 37°C 下培养转化子,我们优化了该系统的筛选过程。该系统被成功用于敲除 JS0135ΔnanH::KanR 中的 KanR 盒,在最后一轮筛选中,敲除效率达到了 90%。为了证实温度变化是一个关键因素,我们继续在 CF7066 菌株中敲除 nanH 和 apd 基因。基因敲除效率高达 100%,最短筛选时间仅为四天。敲除 nanH 基因会显著降低菌株的生长活力,而敲除 apd 基因则会使粘附率提高约 56%。此外,我们还观察到,重组基因在转化株中的表达量在 30℃ 比 37℃ 高,其中 recC 基因上调了约 7 倍。相比之下,野生型菌株重组基因的表达在 30℃ 和 37℃ 之间几乎没有差异。这种差异可能是由于 30℃ 时目的质粒的拷贝数增加,从而导致重组基因的表达增强:总之,这种新开发的寄生虫基因敲除系统是推进寄生虫研究的重要工具。
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A marker-free genetic manipulation method for Glaesserella parasuis strains developed by alternately culturing transformants at 37°C and 30°C.

Background: Glaesserella parasuis (G. parasuis) is the causative agent of Glässer's disease, which causes significant economic losses in the swine industry. However, research on the pathogenesis of G. parasuis has been hampered by the lack of a simple and efficient marker-free knockout system.

Results: In this study, a marker-free knockout system was developed for G. parasuis using a temperature-sensitive vector. By alternating the incubation of transformants at 30°C and 37°C, we optimized the screening process for this system. The system was successfully applied to knockout the KanR cassette from JS0135ΔnanH::KanR, achieving a knockout efficiency of 90% in the final round of screening. To confirm that temperature variation was a key factor, we proceeded with knocking out the nanH and apd genes in the CF7066 strain. The knockout efficiency reached up to 100%, with the shortest screening time being only four days. The knockout of the nanH gene resulted in a significant reduction in the growth vitality of the strains, while the knockout of the apd gene led to an approximate 56% improvement in the adhesion rate. Additionally, we observed that the expression of recombinant genes in transformants was higher at 30℃ than at 37℃, with the recC gene being upregulated approximately 7-fold. In contrast, there was almost no difference in the expression of recombinant genes between 30℃ and 37℃ in the wild-type strains. This discrepancy was likely due to an elevated copy number of target plasmids at 30℃, which may have resulted in the enhanced expression of recombinant genes.

Conclusions: In conclusion, this newly developed gene knockout system for G. parasuis presents a valuable tool for advancing research on this organism.

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来源期刊
BMC Biotechnology
BMC Biotechnology 工程技术-生物工程与应用微生物
CiteScore
6.60
自引率
0.00%
发文量
34
审稿时长
2 months
期刊介绍: BMC Biotechnology is an open access, peer-reviewed journal that considers articles on the manipulation of biological macromolecules or organisms for use in experimental procedures, cellular and tissue engineering or in the pharmaceutical, agricultural biotechnology and allied industries.
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