肖熙鸥 林文秋 陈卓 邹春香 金辉 邹华芬

摘  要：利用標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

中图分类号：S432.4      文献标识码：A

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

XIAO Xiou1，2，3， 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

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 didnt 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 of R. 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.

Keywords： Ralstonia solanacearum; GFP marker; infection dynamic; pBBR1MCS2-Tac-EGFP vector

DOI： 10.3969/j.issn.1000-2561.2021.06.027

青枯雷尔氏菌（Ralstonia solanacearum）寄主范围广泛，能够引起200多种植物感染青枯病[1]。青枯病是茄子和马铃薯等茄科作物生产中主要的病害之一，该病害能造成严重的损失，甚至可以导致绝收。在我国，除西藏、黑龙江和内蒙古外，其余省份均有青枯菌的报道，尤其是在热带和亚热带地区，青枯病危害更为严重[2]。到目前为止，尚无有效的方法对其进行防控。而研究青枯菌的致病机理对防控青枯病具有重要意义。利用标记基因追踪病原菌在植物体内的入侵、定殖等侵染途径，是研究病原菌致病机理的有效手段和方法[3-7]。目前在R. solanacearum中使用的标记基因有GFP[8-10]、LUX[9， 12]和GUS[13]等。标记基因在R. solanacearum中的表达主要有2种方式，一种是利用Tn5转座子……