越南伯克霍尔德氏菌B418的红色荧光蛋白标记及功能稳定性

doi:10.3976/j.issn.1002-4026.2023.01.007

山东科学 ›› 2023, Vol. 36 ›› Issue (1): 51-57.doi: 10.3976/j.issn.1002-4026.2023.01.007

越南伯克霍尔德氏菌B418的红色荧光蛋白标记及功能稳定性

刘敏敏¹(), 吴远征², 扈进冬², 李纪顺², 王燕¹, 杨合同¹^,²^,^*()

1.齐鲁工业大学(山东省科学院) 生物工程学院,山东济南 250353
2.齐鲁工业大学(山东省科学院) 山东省科学院生态研究所山东省应用微生物重点实验室,山东济南 250103

收稿日期:2022-01-14 出版日期:2023-02-20 发布日期:2023-02-08
通信作者: *杨合同,研究员,研究方向为生防微生物。E-mail: yanght@sdas.org。
作者简介:刘敏敏(1996—),女,硕士研究生,研究方向为越南伯克霍尔德氏菌生防机理。E-mail:liuminmin0123@163.com。
基金资助:
山东省省自然科学基金(ZR2021MC085);山东省重点研发计划(重大科技创新工程)(2019JZZY010718);山东省重点研发计划(国际科技合作)(2019GHZ003);山东省科学院国际科技合作项目(2019GHPY05)

Burkholderia vietnamiensis B418 labeled with red fluorescent protein and its functional stability

LIU Minmin¹(), WU Yuanzheng², HU Jindong², LI Jishun², WANG Yan¹, YANG Hetong¹^,²^,^*()

1. School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
2. Shandong Provincial Key Laboratory of Applied Microbiology, Ecology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China

Received:2022-01-14 Online:2023-02-20 Published:2023-02-08

摘要/Abstract

摘要：

利用氨基纳米黏土介导的转化方法,应用红色荧光蛋白DsRed标记生防细菌越南伯克霍尔德氏菌Burkholderia vietnamiensis B418并研究标记菌株的功能稳定性。将带有DsRed基因的重组质粒pGEX-4T-1-DsRed转化导入B418菌株中,采用细菌培养法、继代培养法和平板对峙培养法检测标记菌株的功能稳定性。通过氨基纳米黏土转化体系成功获得了荧光标记菌株B418-DsRed,与野生菌株B418相比,其生长曲线和形态学特征基本一致。继代培养10代后,标记菌株在共聚焦显微镜下呈现亮红色荧光,能够提取到外源质粒pGEX-4T-1-DsRed,并通过聚合酶链式反应扩增获得荧光基因序列DsRed,证明标记菌株具有较好的遗传稳定性。平板对峙培养试验显示,标记菌株B418-DsRed对尖孢镰孢菌和大丽轮枝菌抑制率分别为55.25%和67.55%,与野生菌株B418无显著性差异,表明外源质粒的导入未影响菌株的抑菌能力。以上结果表明通过氨基纳米黏土介导成功实现了伯克霍尔德氏菌B418的红色荧光蛋白标记,构建的标记菌株B418-DsRed可用于该生防菌在植物根际的定殖及与病原菌的互作研究。

关键词: 越南伯克霍尔德氏菌, 红色荧光蛋白, 氨基纳米黏土, 功能稳定性

Abstract:

This study aims to label the biocontrol agent Burkholderia vietnamiensis B418 with the red fluorescent protein DsRed and investigate the functional stability of the labeled strain. The recombinant plasmid pGEX-4T-1-DsRed containing the DsRed gene was transformed into B418 by the aminoclay-mediated transformation system. Germicul ture, subculture, and dual-culture techniques were used to determine the functional stability of the labeled strain B418-Ds Red.The growth curve and morphological characteristics of the strain B418-DsRed after aminoclay transformation were essentially the same as the wild strain B418. After ten generations of subculturing, bright red fluorescence could still be observed under a confocal microscope and the DsRed gene could be detected by polymerase chain reaction, after plasmid extraction from the strain B418-DsRed, which showed its good genetic stability. Dual-culture confrontation assay revealed that the inhibition rates of the strain B418-DsRed against Fusarium oxysporum and Verticillium dahliae were 55.25% and 67.55%, respectively, with no significant difference when compared to the wild strain B418. It was indicated that the introduction of a foreign plasmid showed no impact on their antifungal activity. Results demonstrated that the red fluorescent protein labeling of the strain B418 was successfully achieved by an aminoclay-mediated transformation system and the labeled strain B418-DsRed with stable function could be used in the future for the colonization of the strain B418 and interaction with pathogens in the plant rhizosphere.

Key words: Burkholderia vietnamiensis, DsRed, aminoclay, functional stability

中图分类号:

S476.1

刘敏敏, 吴远征, 扈进冬, 李纪顺, 王燕, 杨合同. 越南伯克霍尔德氏菌B418的红色荧光蛋白标记及功能稳定性[J]. 山东科学, 2023, 36(1): 51-57.

LIU Minmin, WU Yuanzheng, HU Jindong, LI Jishun, WANG Yan, YANG Hetong. Burkholderia vietnamiensis B418 labeled with red fluorescent protein and its functional stability[J]. Shandong Science, 2023, 36(1): 51-57.

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菌株	尖孢镰孢菌		大丽轮枝菌
菌株	菌落直径/cm	抑菌率/%	菌落直径/cm	抑菌率/%
对照CK	8.47±0.12 ^a		8.32±0.09 ^a
B418-DsRed	3.79±0.07^b	55.25	2.70±0.05^b	67.55
B418	3.53±0.10^c	58.32	2.79±0.05^b	66.47

越南伯克霍尔德氏菌B418的红色荧光蛋白标记及功能稳定性

Burkholderia vietnamiensis B418 labeled with red fluorescent protein and its functional stability

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