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浙江大学学报(农业与生命科学版)  2021, Vol. 47 Issue (5): 566-576    DOI: 10.3785/j.issn.1008-9209.2021.02.221
植物保护     
胡萝卜软腐果胶杆菌lux发光菌株的构建和应用
钟灵坤(),徐翠虹,黄泽铭,安千里,梁岩()
浙江大学农业与生物技术学院生物技术研究所/农业农村部作物病虫分子生物学重点实验室,杭州 310058
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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摘要:

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    
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.

Key words: Pectobacterium carotovorum    luciferase    bioluminescence    luxCDABE element    soft rot disease
收稿日期: 2021-02-22 出版日期: 2021-10-27
CLC:  S 436.31  
基金资助: 国家重点研发计划(2018YFD1000800);国家自然科学基金(31622006);浙江省重点研发计划(2017C02002)
通讯作者: 梁岩     E-mail: 21926934@qq.com;yanliang@zju.edu.cn
作者简介: 钟灵坤(https://orcid.org/0000-0001-8226-4488),Tel:+86-28-83301873,E-mail:21926934@qq.com
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钟灵坤
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引用本文:

钟灵坤,徐翠虹,黄泽铭,安千里,梁岩. 胡萝卜软腐果胶杆菌lux发光菌株的构建和应用[J]. 浙江大学学报(农业与生命科学版), 2021, 47(5): 566-576.

Lingkun ZHONG,Cuihong XU,Zeming HUANG,Qianli AN,Yan LIANG. Construction and application of luminescent strain of Pectobacterium carotovorum harboring luxCDABE operon. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(5): 566-576.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.02.221        http://www.zjujournals.com/agr/CN/Y2021/V47/I5/566

基因

Gene

引物名称

Primer name

引物序列(5′→3′)

Primer sequence (5′→3′)

产物长度

Product length/bp

luxCDABEluxFCCGGAATTCATGACTAAAAAAATTTCATTCAT (EcoRⅠ)5 816
luxRCGCGGATCCATCAACTATCAAACGCTTC (BamHⅠ)
luxCluxCFATTTCATTCATTATTAACGGCCAGG588
luxCRCGGATGATTAGGGTCTACATCAATAAAACT
16S rRNA27FGAGAGTTTGATCCTGGCTCAG1 439
1492RTACGGCTACCTTGTTACGAC
表1  引物序列信息
图1  重组质粒图谱
图2  重组质粒的电泳检测M: 8000 DNA分子标志物。
图3  luxC基因的PCR产物电泳图M:2000 DNA分子标志物。
图4  LMG2404-LUX发光成像及菌落形态A.光信号成像(色柱表示光信号强度:+,强;-,弱); B. 菌落形态;C.单个菌落放大图。
图5  LMG2404-LUX生长曲线、生物膜形成以及游动性检测A. LMG2404-LUX生长曲线;B. LMG2404-LUX生物膜特性;C. LMG2404-LUX游动性。CK:空白培养基。
图6  连续继代培养及LMG2404-LUX衰老对光信号的影响A.连续继代培养对LMG2404-LUX光信号的影响(色柱表示光信号强度:0,最弱;10,最强);B. LMG2404-LUX衰老与光信号强度的关系。
图7  温度和pH对LMG2404-LUX光信号强度的影响短栅上的不同小写字母表示在P<0.05水平差异有统计学意义。
图8  LMG2404-LUX菌的光信号强度与菌液浓度的相关性
图9  LMG2404-LUX侵染拟南芥后不同天数的叶片病情指数0~4表示不同病情指数,0级代表发病最轻,4级代表发病最重。CK:空白培养基。
图10  LMG2404-LUX侵染拟南芥后的光信号强度检测A.拟南芥叶片接种LMG2404-LUX后1~3 d的光信号(短栅上的不同小写字母表示在相同天数不同处理间在P<0.05水平差异有统计学意义);B.拟南芥接种LMG2404-LUX后1 d的发光成像(红色箭头表示水渍状病斑;色柱表示光信号强度:+,强;-,弱);C.拟南芥接种LMG2404-LUX后36 h内的光信号。CK:空白培养基。
图11  LMG2404-LUX侵染白菜、胡萝卜和马铃薯后的症状及光信号检测A. LMG2404-LUX分别侵染白菜、胡萝卜和马铃薯后的发光成像;B~D. LMG2404-LUX分别侵染白菜、胡萝卜和马铃薯后的病斑面积;E~G. LMG2404-LUX分别侵染白菜、胡萝卜和马铃薯后的光信号强度。CK:空白培养基。
图12  luxCDABE在大肠埃希菌和农杆菌中的表达色柱表示光信号强度(+,强;-,弱)。
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