sacB介导的绿针假单胞菌YL-1遗传操作方法
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摘要
绿针假单胞菌Pseudomonas chlororaphis YL-1是从大豆根围分离获得的,对多种病原细菌和病原真菌有较强抑制作用。为了探明绿针假单胞菌YL-1生防相关基因的功能,本研究以嗜铁素转录调控因子PvdS编码基因为对象,建立了一套基于负选择标记基因sacB的绿针假单胞菌无标记基因敲除技术,构建重组质粒p EX18-pvdS,通过改良的细菌接合转移技术将重组质粒导入野生型菌株YL-1中,利用同源重组技术获得缺失突变株ΔpvdS。生防相关性状研究结果表明,与野生型菌株YL-1相比,突变株ΔpvdS泳动能力和生长能力未发生改变,但是群集运动能力显著下降。同时突变株ΔpvdS合成嗜铁素的能力也显著下降,pvdS基因互补后突变株能恢复合成嗜铁素的功能。本文结果表明,已成功建立了适用于YL-1的基因定向敲除技术和功能基因互补体系,为深入研究YL-1的生防机制奠定了重要基础。
Strain YL-1 was isolated from soybean root tips and identified as Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant phytopathogens that are economically important in agriculture. In order to explore the function of its biocontrol-associated genes, an efficient genetic manipulation system was established in this study. The Bacillus subtilis sacB gene, whose product encodes levansucrase that is toxic to Gram-negative bacteria in the presence of sucrose, was considered as a counterselection marker in the system. We selected a well-characterized pvdS as a representative example to generate an in-frame deletion mutant, because the product of pvdS encodes a sigma factor that is known to control pyoverdine biosynthesis at transcription level. A sacB-containing suicide-vector, pEX18 was used, in which both the upstream and downstream homolog fragment of pvdS was cloned, creating a recombinant plasmid, pEX18-pvdS. The recombinant plasmid was transformed into wild type strain YL-1 via an optimized bacterial conjugal approach. Via a double-crossover homologous recombinant approach, an in-frame deletion mutant of pvdS, named as ΔpvdS was generated and validated. Compared to the wild type, this mutant exhibited equal swimming motility and growth capacity, but a significant decrease in swarming motility and pyoverdine production.The function of pyoverdine production in the ΔpvdS could be rescued by introducing a plasmid-borne pvdS into this mutant. Together, our studies established an effective system for both in-frame gene deletion and complementation in YL-1, which facilitates to uncover the biocontrol mechanisms of YL-1 in the future.
Strain YL-1 was isolated from soybean root tips and identified as Pseudomonas chlororaphis. This strain showed broad-spectrum antibacterial and antifungal activities against plant phytopathogens that are economically important in agriculture. In order to explore the function of its biocontrol-associated genes, an efficient genetic manipulation system was established in this study. The Bacillus subtilis sacB gene, whose product encodes levansucrase that is toxic to Gram-negative bacteria in the presence of sucrose, was considered as a counterselection marker in the system. We selected a well-characterized pvdS as a representative example to generate an in-frame deletion mutant, because the product of pvdS encodes a sigma factor that is known to control pyoverdine biosynthesis at transcription level. A sacB-containing suicide-vector, pEX18 was used, in which both the upstream and downstream homolog fragment of pvdS was cloned, creating a recombinant plasmid, pEX18-pvdS. The recombinant plasmid was transformed into wild type strain YL-1 via an optimized bacterial conjugal approach. Via a double-crossover homologous recombinant approach, an in-frame deletion mutant of pvdS, named as ΔpvdS was generated and validated. Compared to the wild type, this mutant exhibited equal swimming motility and growth capacity, but a significant decrease in swarming motility and pyoverdine production.The function of pyoverdine production in the ΔpvdS could be rescued by introducing a plasmid-borne pvdS into this mutant. Together, our studies established an effective system for both in-frame gene deletion and complementation in YL-1, which facilitates to uncover the biocontrol mechanisms of YL-1 in the future.
引文
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[16]邹丽芳,周丹,刘之洋,等.水稻黄单胞菌致病相关基因插入突变体系的建立[J].植物病理学报,2012,42(2):176-185.
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[18]赵龙华,杨维青.细菌群集运动与生物被膜和耐药性的关系[J].国际检验医学杂志,2011,32(17):1986-1988.
[19]Gao M,Song H,Liu X,et al.Improved quorum sensing capacity by culturing Vibrio harveyi in microcapsules[J].Journal of Bioscience and Bioengineering,2016,121(4):406-412.
[20]Guo Y Z,Liu S T,Tang X,et al.Role of c-di-GMP in anammox aggregation and systematic analysis of its turnover protein in Candidatus Jettenia caeni[J].Water Research,2017,113:181-190.
[2]刘邮洲,Lu S,Baird S M,等.绿针假单胞菌YL-1抗细菌活性相关基因的克隆和分析[J].植物病理学报,2015,45(3):307-316.
[3]Liu Y Z,Lu S E,Braid S,et al.Draft genome sequence of Pseudomonas chlororaphis YL-1,a biocontrol strain suppressing plant microbial pathogens[J].Genome Announcements,2014,2(1):1-2.
[4]张勇.水稻白叶枯病菌和条斑病菌对噻枯唑和链霉素的抗药性机理研究[D].南京:南京农业大学,2011.
[5]Reyret J M,Pelicic V,Gicquel B,et al.Counterselectable markers:untapped tools for bacterial genetics and pathogenesis[J].Infection and Immunity,1998,66(9):4011.
[6]Lu S E,Scholz-Schroeder B K,Gross D C.Characterization of the salA,syrF,and syrG regulatory genes located at the right border of the syringomycin gene cluster of Pseudomonas syringae pv.syringae[J].Molecular Plant-Microbe Interactions,2002,15:43-53.
[7]刘轶儒,徐菲菲,钱国良,等.产酶溶杆菌rpfG基因的克隆与功能分析[J].南京农业大学学报,2013,36(2):45-50.
[8]萨姆布鲁克J,拉塞尔D W.分子克隆试验指南[M].北京:科学出版社,2002.
[9]张惠展.途径工程-第三代基因工程[M].北京:中国轻工业出版社,2002.
[10]Cornelia R,Nathalie G,Laurent M,et al.Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1[J].Microbiology,2002,148:923-932.
[11]赵龙飞,徐亚军,常佳丽.轮纹病菌拮抗性大豆根瘤内生菌的筛选、抗性和促生作用[J].中国生物防治学报,2016,32(3):396-405.
[12]Liu Y Z,Baird S M,Qiao J Q,et al.SecG is required for antibiotic activities of Pseudomonas sp.YL23 against Erwinia amylovora and Dickeya chrysanthemi[J].Journal of Basic Microbiology,2015,55:617-624.
[13]Visaggio D,Pasqua M,Bonchi C,et al.Cell aggregation promotes pyoverdine-dependent iron uptake and virulence in Pseudomonas aeruginosa[J].Frontiers in Microbiology,2015,6:1-11.
[14]于非非,余祥勇,于海英,等.异油酸含量对铜绿假单胞菌群集运动能力的影响[J].微生物学报,2015,55(1):1600-1607.
[15]张霞,张杰,李国勋,等.绿色荧光蛋白标记荧光假单胞菌P303及其生存能力检测[J].植物保护学报,2005,32(3):280-286.
[16]邹丽芳,周丹,刘之洋,等.水稻黄单胞菌致病相关基因插入突变体系的建立[J].植物病理学报,2012,42(2):176-185.
[17]陈鑫,杨振飞,朱芮,等.稀有放线菌小单孢菌与大肠杆菌接合转移体系的构建[J].华中农业大学学报,2015,34(4):80-83.
[18]赵龙华,杨维青.细菌群集运动与生物被膜和耐药性的关系[J].国际检验医学杂志,2011,32(17):1986-1988.
[19]Gao M,Song H,Liu X,et al.Improved quorum sensing capacity by culturing Vibrio harveyi in microcapsules[J].Journal of Bioscience and Bioengineering,2016,121(4):406-412.
[20]Guo Y Z,Liu S T,Tang X,et al.Role of c-di-GMP in anammox aggregation and systematic analysis of its turnover protein in Candidatus Jettenia caeni[J].Water Research,2017,113:181-190.