| 黄金玲,吴雨霜,唐慧全,等.花生青枯菌GFPuv标记菌株的构建和致病性分析[J].广东农业科学,2024,(10-11):- |
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花生青枯菌GFPuv标记菌株的构建和致病性分析 |
| Construction and Pathogenicity Analysis of a GFPuv-labeled Peanut Ralstonia solanacearum Strain |
| 投稿时间：2024-07-28 修订日期：2024-09-05 |
| DOI： |
| 中文关键词: 青枯菌 绿色荧光蛋白 标记菌株 整合位点 致病性分析 |
| 英文关键词: Ralstonia solanacearum GFPuv Labeled strain Integration site Pathogenicity analysis |
| 基金项目:广东省基础与应用基础研究项目(2022A1515110018)；仲华基因科技产业学院大学生创新创业训练计划项目(ZHCY2023001)；大湾区生物育种产业学院大学生产业技术训练计划项目(NSCY2023001)； |
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| 中文摘要: |
| 【目的】本研究旨在构建带有GFPuv标记的花生青枯菌，以实时观察其在宿主植物内的侵染路径，并确定适用于外源基因整合的基因组位点。【方法】通过对青枯菌PeaFJ1菌株的全基因组分析，选定外源基因的插入位点。利用pK18mobsacB自杀质粒，应用同源重组双交换技术将GFPuv基因整合到PeaFJ1菌株基因组的指定位点。通过荧光显微镜在紫外光激发下评估GFPuv标记菌株的荧光表现，通过生长速率和致病力测定评估标记菌株的功能活性。【结果】GFPuv基因成功整合至PeaFJ1的IR3区域。GFPuv标记菌株在紫外光下显现强烈绿色荧光，在液体培养基中生长96 h后的OD600值达到的1.5以上，在固体培养基上生长48 h后达到0.5以上，生长3 d的菌落直径达到1.0~1.5 mm，在侵染花生后11 d使植物萎蔫死亡率达到100%，这些特征与野生型菌株无显著差异。【结论】成功构建GFPuv插入基因组IR3区域的标记青枯菌PeaFJ1-GFPuv菌株，在生物学特性和致病性方面与野生型无显著差异，证明IR3区域为PeaFJ1基因回补的有效位点。GFPuv标记菌株在紫外光下显现强烈绿色荧光，可用于观察青枯菌侵染路径。 |
| 英文摘要: |
| 【Objective】This study aimed to construct a GFPuv-labeled peanut Ralstonia solanacearum, that was used to observe the infection trace in host plants in real-time. Meanwhile, genomic integration sites suitable for exogenous gene were identified.【Method】Through whole-genome analysis of R. solanacearum PeaFJ1, the insertion site for exogenous gene was selected. The suicide plasmid pK18mobsacB and homologous recombination double exchange technology was employed to integrate the GFPuv gene into the designated site of PeaFJ1 genome. Expression of GFPuv was assessed under UV light with fluorescence microscopy. Growth rate and pathogenicity determination were performed to evaluated the functional activity of the GFPuv-labeled strain. 【Results】The GFPuv gene was successfully integrated into the IR3 region of PeaFJ1 genome. The GFPuv-labeled strain exhibited strong green fluorescence under UV light. The OD600 value reached more than 1.5 after 96 h growth in liquid medium, and more than 0.5 after 48 h growth in solid medium. The diameter of a single colony reached 1~1.5 mm. At 11 days after infection, the death rate of plant reached 100%. All these characteristics were not significantly different from those of wild type strains.【Conclusion】The GFPuv-labeled R. solanacearum PeaFJ1 strain was successfully constructed. demonstrated biological characteristics and pathogenicity consistent with the wild type. There was no significant difference in biological characteristics and pathogenicity between the GFPuv-labeled strain and the wild type, indicating that the IR3 region is an effective site for gene complementation. confirming that the GFPuv insertion did not affect the strain pathogenicity. The GFPuv- labeled strain showed strong green fluorescence under UV light, which could be used to observe the infection trace of R. solanacearum. |
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