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

doi:10.3969/j.issn.1000-2561.2021.06.027

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

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. 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. 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/Abstract

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

中图分类号:

S432.4

肖熙鸥, 林文秋, 陈卓, 邹春香, 金辉, 邹华芬. pBBR1MCS2-Tac-EGFP广宿主载体适宜标记Ralstonia solanacearum[J]. 热带作物学报, 2021, 42(6): 1700-1705.

XIAO Xi’ou, LIN Wenqiu, CHEN Zhuo, ZOU Chunxiang, JIN Hui, ZOU huafen. Wide-host Vector pBBR1MCS2-Tac-EGFP Suitable for the Labeling of Ralstonia solanacearum[J]. Chinese Journal of Tropical Crops, 2021, 42(6): 1700-1705.

图/表 8

图1 广宿主载体pBBR1MCS2-Tac-EGFP图谱

Fig. 1 Map of pBBR1MCS2-Tac-EGFP Vector

图2 GMI1000-Tac-EGFP菌株PCR检测 M：DL2000 maker;+：pBBR1MCS2-Tac-EGFP质粒;1~5：GMI1000-Tac-EGFP克隆;-：GMI1000。

Fig. 2 PCR detection of GMI1000-Tac-EGFP strain M: DL2000 maker; +: pBBR1MCS2-Tac-EGFP plasmid; 1-5: GMI1000-Tac-EGFP clone; -: GMI1000.

图3 GMI1000-Tac-EGFP荧光检测 GMI1000-Tac-EGFP在440 nm波长下发出绿色荧光;W：GMI1000;G：GMI1000-Tac-EGFP。

Fig. 3 Detection of GFP fluorescence of GMI1000-Tac-EGFP GMI1000-Tac-EGFP was emission GFP fluorescence under 440 nm excitation light; W: GMI1000; G: GMI1000- Tac-EGFP.

图4 GMI1000-Tac-EGFP不同继代次数荧光强度

Fig. 4 Fluorescence intensity between different generation of GMI1000-Tac-EGFP

图5 EGFP荧光强度与GMI1000-Tac-EGFP数量呈线性关系

Fig. 5 Correlation of fluorescence intensity and R. solanacearumcell number

图6 马铃薯茎部R. solanacearum克隆数

Fig. 6 GMI1000-Tac-EGFP cell number of potato stem

图7 GMI1000-Tac-EGFP和GMI1000接种6 d马铃薯叶片出现萎蔫症状

Fig. 7 Potato wilt 6 d after inoculation with GMI1000- Tac-EGFP and GMI1000

图8 利用GMI1000-Tac-EGFP分析R. solanacearum侵染马铃薯动态变化过程 A：R. solanacearum侵染马铃薯动态变化过程;B：接种后1 d R. solanacearum已经进入到马铃薯根部;bar=50 μm。

Fig. 8 Analysis of dynamic infection progress ofR. solanacearum in ptotato A: Dynamic infection progress ofR. solanacearum in ptotato; B: R. solanacearum inoculation the potato root after 1 d inoculation. bar=50 μm.

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

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

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