四株革兰氏阴性细菌的红绿色荧光蛋白标记及其生物学特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0550

摘要/Abstract

摘要：

【目的】构建广谱复制型荧光蛋白质粒，并对革兰氏阴性细菌标记以评价荧光蛋白质粒适用性。【方法】将gfp、mCherry和组成型启动子PpsbA克隆到质粒pBBR1MCS2'以构建表达载体pBBR1MCS2'-PpsbAGFP和pBBR1MCS2'-PpsbAmCherry，通过接合转移将上述载体分别导入茄科劳尔氏菌（Ralstonia solanacearum）EP1、胡萝卜果胶杆菌（Pectobactererium carotovorum subsp. carotovorum）WB、肺炎克雷伯氏菌（Klebsiella Pneumoniae）JF和洋葱伯克霍尔德氏菌（Burkholderia cepacia）1-2中，观察菌落及细胞荧光表现，检测其生长曲线和质粒保持率，并验证相关功能。【结果】荧光蛋白表达载体pBBR1MCS2'-PpsbAGFP和pBBR1MCS2'-PpsbAmCherry被成功构建，并成功地利用绿色和红色荧光蛋白标记了上述4株革兰氏阴性细菌。荧光蛋白标记菌的菌落及细胞个体均有对应的荧光发出，生长速率与野生型菌株一致。然而，荧光蛋白质粒在此4株细菌中的稳定性存在较大差异：绿色和红色荧光蛋白质粒在JF菌株中的稳定性最高，无选择压力继代培养40 h后的质粒持有率分别达90.33%和94.67%，在WB和EP1菌株中次之，在1-2菌株中的稳定性均最差，分别在培养5 h和25 h后就检测不到荧光。此外，荧光标记的1-2菌株溶磷活性和WB致病性与各自野生型菌株无显著差异。【结论】成功构建2种荧光蛋白表达载体，并对 R. solanacearum EP1 等4株不同革兰氏阴性细菌的荧光蛋白标记。其在质粒保持率中，绿色和红色荧光蛋白在1-2菌株中稳定性较差，在其余3株菌株中均能稳定表达，为后续为革兰氏阴性细菌的标记及其相关功能研究提供重要的研究材料。

关键词: 革兰氏阴性细菌, 荧光蛋白标记, 广谱复制型, 接合转移, 质粒保持率

Abstract:

【Objective】To construct broad host range replication type fluorescent protein plasmid and to evaluate the applicability of fluorescent protein plasmid by labeling four strains of Gram-negative bacteria. 【Method】gfp, mCherry and the constitutive promoter PpsbA were cloned into the plasmid pBBR1MCS2' in order to construct the expression vector pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry, and the expression vectors were introduced into Ralstonia solanacearum EP1, Pectobactererium carotovorum subsp. carotovorum WB, Klebsiella Pneumoniae JF and Burkholderia cepacia 1-2 by conjugative transfer. Their fluorescence performance of colonies and cells was observed, growth curves and plasmid retention rates were detected, and relevant functions were verified. 【Result】The fluorescent protein expression vectors pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry were successfully constructed, and the four Gram-negative bacteria were successfully labeled with green and red fluorescent proteins. The colonies and individual cells of the fluorescent protein-labeled bacteria emitted corresponding fluorescence, and the growth rate was consistent with that of the wild-type strains. However, the stability of the fluorescent protein plasmids varied greatly among the four strains. The green and red fluorescent protein plasmids were the most stable in the strain JF, with plasmid retention rates of 90.33% and 94.67%, respectively after 40 h of incubation without selective pressure; followed by strain WB and EP1, and the worst in strain 1-2, with fluorescence not detected after 5 and 25 h of incubation, respectively. In addition, there was no significant difference in the pathogenicity between fluorescently labeled WB and wild-type strain WB. 【Conclusion】 Two fluorescent protein expression vectors are successfully constructed and introduced into four different Gram-negative bacterial strains, including R. solanacearum EP1. In the plasmid retention rate, green and red fluorescent proteins are less stable in strain 1-2, and are stably expressed in the remaining three strains. These vectors provide important research materials for the subsequent study of labelling Gram-negative bacteria and investigating their related functions.

Key words: Gram-negative bacteria, broad host range replication type, fluorescent protein labeling, conjugative transfer, plasmid retention rate

吴启叶, 卢丽娜, 刘迎龙, 平媛, 何鹏搏, 何月秋, 吴毅歆, 何鹏飞. 四株革兰氏阴性细菌的红绿色荧光蛋白标记及其生物学特性研究[J]. 生物技术通报, 2024, 40(12): 239-247.

WU Qi-ye, LU Li-na, LIU Ying-long, PING Yuan, HE Peng-bo, HE Yue-qiu, WU Yi-xin, HE Peng-fei. Four Strains of Gram-negative Bacteria Labeled with Red and Green Fluorescent Proteins and Their Biological Characterization[J]. Biotechnology Bulletin, 2024, 40(12): 239-247.

图/表 8

表1 构建载体所用引物

Table 1 Primers used for cloning（vector construction）

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
PpsbA-F	CGCGC GAATTCGAGCTCGGT（EcoR I）
PpsbA-R	ATGT AGATCTCCTTCTTAAAGT（Bgl II）
PpsbAG-R	AGA GGATCCGTCGACTTATTATTTGTATAGTTCAT（BamH I、Sal I）
mCherry-F	GCG AGATCTACATATGGTGAGCAAGGGCGAGGA（Bgl II）
mCherry-R	GA GGATCCGTCGACTTACTTGTACAGCTCGTCCAT（BamH I、Sal I）

图1 绿色荧光表达质粒pBBR1MCS2'- PpsbAGFP的构建流程图

Fig. 1 Flow chart of pBBR1MCS2'-PpsbAGFP expressing green fluorescent protein

图2 pBBR1MCS2'- PpsbAGFP和pBBR1MCS2'- Pps-bAmCherry载体构建结果 A：PpsbA（泳道2）、mCherry（泳道3）、PpsbAmCherry（泳道4）、PpsbAGFP（泳道5）的电泳检测；M：DNA marker 2000；泳道1：CK；B：pBBR1MCS2'-PpsbAGFP和pBBR1MCS2'-PpsbAmCherry的酶切验证；M：DNA marker 250II；1：pBBR1MCS-2'酶切产物；2：pBBR1MCS-2'质粒；3：pBBR1MCS2'-PpsbAGFP酶切结果；4：pBBR1MCS2'-PpsbAmCherry酶切结果

Fig. 2 Confirmation Result of pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry vector construction A: Gel electrophoretic visualization of PpsbA（lane 2）, mCherry（lane 3）, PpsbAmCherry（lane 4）, PpsbAGFP（lane 5）. M: DNA marker 2000; lane 1: CK. B: Restriction analysis of pBBR1MCS2'-PpsbAGFP and pBBR1MCS2'-PpsbAmCherry. M: DNA marker 250II; lane 1: restriction products of pBBR1MCS-2'; lane 2: pBBR1MCS-2' plasmid; lane 3: restriction results of pBBR1MCS2'-PpsbAGFP; lane 4: restriction results of pBBR1MCS2'-PpsbAmCherry

图3 荧光蛋白标记菌的荧光表现（比例尺：3 μm）

Fig. 3 Images of fluorescence proteins-tagged bacteria（Scale bar: 3 μm）

图4 R. solanacearum EP1（A）、P. carotovorum WB（B）、K. pneumoniae JF（C）和B. cepacia 1-2（D）及其荧光蛋白标记菌的生长曲线

Fig. 4 Growth curves of fluorescent proteins tagged R. solanacearum EP1（A）, P. carotovorum WB（B）, K. pneumoniaeJF（C）, and B. cepacia 1-2（D）

图5 绿色（A）和红色（B）荧光蛋白质粒在供试菌株中的质粒保持率

Fig. 5 Plasmid retention rates of green（A）and red （B）fluorescent protein plasmidin the tested strains

图6 B. cepacia 1-2及其标记菌株溶磷活性差异（第5天） A：B. cepacia 1-2；B：B. cepacia 1-2-PpsbAGFP；C：B. cepacia 1-2-PpsbAmCherry；D：溶磷圈直径（D）与菌落直径（d）的比值

Fig. 6 Differences in phosphate-solubilizing activity of B. cepacia 1-2 and labelled strains（day 5） Pathogenicity of P. carotovorum WB and protein fluorescent labelled strain on Chinese cabbage（2 d post-inoculation, scale bar: 1 cm） A: B. cepacia 1-2; B: B. cepacia 1-2-PpsbAGFP; C: B. cepacia 1-2-PpsbAmCherry;D: ratio of phosphate-solubilizing zone diameter（D）to colony diameter（d）

图7 P. carotovorum WB及其荧光标记菌对大白菜叶柄组织的致病性（接种后2 d，比例尺：1 cm）

Fig. 7 Pathogenicity of P. carotovorum WB and protein fluorescent labelled strain on Chinese cabbage（2 d post-inoculation, scale bar: 1 cm）

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