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热带作物学报 ›› 2021, Vol. 42 ›› Issue (6): 1700-1705.DOI: 10.3969/j.issn.1000-2561.2021.06.027

• 植物保护与生物安全 • 上一篇    下一篇

pBBR1MCS2-Tac-EGFP广宿主载体适宜标记Ralstonia solanacearum

肖熙鸥1,2,3, 林文秋1,2, 陈卓1, 邹春香4, 金辉1,2,*(), 邹华芬1   

  1. 1. 中国热带农业科学院南亚热带作物研究所,广东湛江 524091
    2. 农业农村部热带果树生物学重点实验室,广东湛江 524091
    3. 甘肃农业大学农学院,甘肃兰州 730070
    4. 广州市园林建筑工程公司,广东广州 510180
  • 收稿日期:2020-05-19 修回日期:2020-07-28 出版日期:2021-06-25 发布日期:2021-07-19
  • 通讯作者: 金辉
  • 作者简介:金 辉,E-mail: jh3635315@sina.com
    肖熙鸥(1988—),男,硕士,研究方向:蔬菜育种及生物技术。
  • 基金资助:
    国家自然科学(31872117);中国热带农业科学院基本科研业务费专项资金项目(1630062017014)

Wide-host Vector pBBR1MCS2-Tac-EGFP Suitable for the Labeling of Ralstonia solanacearum

XIAO Xi’ou1,2,3, LIN Wenqiu1,2, CHEN Zhuo1, ZOU Chunxiang4, JIN Hui1,2,*(), ZOU huafen1   

  1. 1. South Subtropical Crop Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, Guangdong 524091, China
    2. Key Laboratory of Tropical Fruit Biology of Ministry of Agriculture & Rural Affairs, Zhanjiang, Guangdong 524091, China
    3. College of Agronomy, Gansu Agricultural University, Lanzhou, Gansu 730070, China
    4. Guagnzhou Landscape Architecture Company, Guangzhou, Guangdong 510180, China
  • Received:2020-05-19 Revised:2020-07-28 Online:2021-06-25 Published:2021-07-19
  • Contact: JIN Hui
  • Supported by:
    中国热带农业科学院基本科研业务费专项资金项目(1630062017016)

摘要:

利用标记基因追踪病原菌在植物体内的入侵和定殖,是研究病原菌-寄主互作的重要手段。本研究利用电转化法将广宿主载体pBBR1MCS2-Tac-EGFP导入青枯雷尔氏菌(Ralstonia solanacearum)GMI1000菌株中,获得了青枯雷尔氏菌带绿色荧光标记的转化子。转接试验结果表明,转化子的抗生素抗性和绿色荧光强度有良好的遗传稳定性。pBBR1MCS2-Tac-EGFP不影响GMI1000菌株的致病力,且EGFP蛋白能够在植物中稳定表达。灌根法接种试验结果表明,病原菌在第1天即完成对根系的侵染,并在第6天扩散至其他组织,随后造成植株萎蔫。研究结果表明所获转化子可用于后续的病原菌侵染机理等方面的研究。

关键词: 青枯雷尔氏菌, 绿色荧光标记, 侵染动态, pBBR1MCS2-Tac-EGFP载体

Abstract:

It is an important method to trace the invasion and colonization of pathogens in plants by marker genes. In the present study, the wide-host vector pBBR1MCS2-Tac-EGFP was transformed into the Ralstonia solanacearum GMI1000 strains by electroporation. Then the GFP expression and the GMI1000 pathogenicity were analyzed. The GMI1000-Tac-EGFP strains emitted GFP fluorescence under the 440 nm excitation light. After the susceptible eggplant was inoculated by wild type GMI1000 and GMI1000-Tac-EGFP, there was no difference between the GMI1000 and the GMI1000-Tac-EGFP in the disease index. The result suggested that GMI1000-Tac-EGFP didn’t influence the pathogenicity of GMI1000. After inoculating eggplants and potatoes by GMI1000-Tac-EGFP, the eggplant root, potato root and potato stem emitted GFP fluorescence under the 440 nm excitation light. The result suggested that GMI1000-Tac-EGFP could express EGFP in the eggplant and potato. To monitor the moment ofR. solanacearum cells in potato, potato was inoculated with GMI1000-Tac-EGFP. The result showed that GMI1000-Tac-EGFP invaded the root 1 d after inoculation, spreaded to the stem 6 d after inoculation. In conclusion, the wide-host vector pBBR1MCS2-Tac-EGFP is suitable for the labeling of R. solanacearum and an important tool to trace the invasion and colonization of R. solanacearum strains in plants.

Key words: Ralstonia solanacearum, GFP marker, infection dynamic, pBBR1MCS2-Tac-EGFP vector

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