Plant protection |
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Construction and application of luminescent strain of Pectobacterium carotovorum harboring luxCDABE operon |
Lingkun ZHONG(),Cuihong XU,Zeming HUANG,Qianli AN,Yan LIANG() |
Institute of Biotechnology, College of Agriculture and Biotechnology/Ministry of Agriculture and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310058, China |
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Abstract Pectobacterium carotovorum is one of the top ten plant bacterial pathogens in the world. In order to facilitate the observation of the pathogen infection process at the early stage and in vivo quantification of bacterial growth without the tissue extraction, we generated a P. carotovorum strain expressing the luxCDABE operon, which encodes a luciferase and enzymes that produce its substrate, thus bacteria that express this operon emit light spontaneously. The luxCDABE operon was cloned into the vector pBBR1MCS2, and the resulting pBBR1MCS2-luxCDABE plasmid was transformed into P. carotovorum subsp. carotovorum LMG2404, namely LMG2404-LUX. Compared with LMG2404, LMG2404-LUX had no significant differences in growth rate, biofilm formation and motility. Luminescent signal intensities of LMG2404-LUX were linearly correlated with the bacterial concentrations and not significantly reduced after successive subculturing for ten times. In addition, those signals were stable at the temperature and pH suitable for bacterial growth. When inoculating plants, LMG2404-LUX and LMG2404 had similar pathogenicity, and the signal intensity of LMG2404-LUX was positively correlated with its infection and proliferation. Collectively, P. carotovorum tagged with luxCDABE could greatly facilitate rapid bacterial quantification and in vivo observation of bacterial infection in host tissues, which provides a new tool for the research on P. carotovorum.
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Received: 22 February 2021
Published: 27 October 2021
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Corresponding Authors:
Yan LIANG
E-mail: 21926934@qq.com;yanliang@zju.edu.cn
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胡萝卜软腐果胶杆菌lux发光菌株的构建和应用
胡萝卜软腐果胶杆菌(Pectobacterium carotovorum)是世界十大植物细菌性病原之一。为了便于观察该病原菌侵染过程以及进行早期活体定量检测,本研究构建了表达luxCDABE基因的胡萝卜软腐果胶杆菌发光菌株。首先构建了含有luxCDABE基因操纵子的重组质粒,然后转入胡萝卜软腐果胶杆菌LMG2404中,命名为LMG2404-LUX。与LMG2404相比,LMG2404-LUX的生长速率、生物膜形成和游动性无明显差异;光信号强度与菌液浓度呈线性相关;在菌适宜生长条件下,光信号不受温度和pH的影响;经过10次继代培养后,光信号强度无明显减弱。LMG2404-LUX接种植物后,其致病性与LMG2404无明显差异,侵染初期的光信号强度与该菌侵染增殖呈线性相关。综上所述,LMG2404-LUX可以用于胡萝卜软腐果胶杆菌的快速定量以及植物体内的活体检测,为该菌的相关研究提供了新的工具。
关键词:
胡萝卜软腐果胶杆菌,
荧光素酶,
生物发光,
luxCDABE元件,
软腐病
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