辣椒溶杆菌Lysobacter capsici X2-3遗传操作体系的建立
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摘要
辣椒溶杆菌(Lysobacter capsici)X2-3是本实验室从小麦根际土壤中分离的对多种植物病原真菌和卵菌有明显拮抗活性的菌株,建立有效的遗传操作体系对了解其基因的功能具有重要意义。本研究以VgrG为目标基因,比较了不同载体对X2-3基因敲除效果,结果发现,载体pEX18GM、pBR325和pK19mob转化X2-3后,均未得到VgrG缺失突变体;而载体pKMS1转化X2-3后,经10%蔗糖二次筛选、PCR和Southern blot验证等,成功得到VgrG缺失突变体;用广宿主载体pBBR1MCS-5构建的VgrG互补突变体,可以恢复VgrG基因的功能。本结果为进一步研究该菌的基因功能提供了技术保障。
Lysobacter capsici X2-3 was isolated by our lab from wheat rhizosphere soil with antimycotic activities to many plant pathogenic fungi and oomycetes. Establishing an effective genetic manipulation system was of great significance for understanding the function of its genes. Using VgrG gene as target, the study compared gene knockout effect of four different vectors on X2-3. The results showed that X2-3 transformed by the vectors pEX18 GM, pBR325 and pK19 mob were not VgrG deletion mutates. However, after transformation of vector pKMS1 into X2-3, VgrG deletion mutants were successfully obtained by 10% sucrose secondary screening, PCR and Southern blot verification. The complemented mutants of VgrG constructed by broad-host vector pBBR1 MCS-5 also restored the function of VgrG gene. This study provided a technical guarantee for the further study on the gene function of the strain X2-3.
Lysobacter capsici X2-3 was isolated by our lab from wheat rhizosphere soil with antimycotic activities to many plant pathogenic fungi and oomycetes. Establishing an effective genetic manipulation system was of great significance for understanding the function of its genes. Using VgrG gene as target, the study compared gene knockout effect of four different vectors on X2-3. The results showed that X2-3 transformed by the vectors pEX18 GM, pBR325 and pK19 mob were not VgrG deletion mutates. However, after transformation of vector pKMS1 into X2-3, VgrG deletion mutants were successfully obtained by 10% sucrose secondary screening, PCR and Southern blot verification. The complemented mutants of VgrG constructed by broad-host vector pBBR1 MCS-5 also restored the function of VgrG gene. This study provided a technical guarantee for the further study on the gene function of the strain X2-3.
引文
[1] Das M M,Haridas M,Sabu A.Biological control of black pepper and ginger pathogens,Fusarium oxysporum,Rhizoctonia solani and Phytophthora capsici,using Trichoderma spp[J].Biocatalysis and Agricultural Biotechnology,2019,1(17):177-183.
[2] Deng Q,Xiao L,Liu Y,et al.Streptomyces avermitilis industrial strain as cell factory for Ivermectin B1a production[J].Synthetic and Systems Biotechnology,2019,4(1):34-39.
[3] Fu Y,Yu Z,Liu S,et al.c-di-GMP regulates various phenotypes and insecticidal activity of Gram-positive Bacillus thuringiensis[J].Frontiers in Microbiology,2018,9:45.
[4] 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.
[5] El-Tarabily K A,Nassar A H,Hardy G E,et al.Plant growth promotion and biological control of Pythium aphanidermatum,a pathogen of cucumber,by endophytic actinomycetes[J].Journal of Applied Microbiology,2009,106(1):13-26.
[6] Zúňiga A,Poupin M J,Donoso R et al.Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN[J].Mol.Plant-Microbe Interact.,2013,26(5):546-553.
[7] 刘朝霞.辣椒溶杆菌X2-3抗菌物质的初步分离及其特性分析[D].泰安:山东农业大学,2016.
[8] Yi J L,Wang J,Li Q,et al.Draft genome sequence of the bacterium Lysobacter capsici X2-3,with a broad spectrum of antimicrobial activity against multiple plant-pathogenic microbes[J].Genome announcements,2015,3 (3):e00589-15.
[9] Qian G,Wang Y,Qian D,et al.Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11[J].World J.Microbiol.Biotechnol.,2012,28(2):549-557.
[10] Qian G,Xu F,Venturi V,et al.Roles of a solo LuxR in the biological control agent Lysobacter enzymogenes strain OH11[J].Phytopathology,2014,104(3):224-231.
[11] Zou L F ,Li Yu R,Chen G Y .A non-marker mutagenesis strategy to generate poly-hrp gene mutants in the rice pathogen Xanthomonas oryzae pv.oryzicola[J].Agricultural Sciences in China,2011,10(8):1139-1150.
[12] Prentki P,Karch F,Iida S,et al.The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication[J].Gene,1981,14(4):289-299.
[13] Katzen F,Becker A,Verónica Ielmini M,et al.New mobilizable vectors suitable for gene replacement in gram-negative bacteria and their use in mapping of the 3' end of the Xanthomonas campestris pv.campestris gum operon[J].Appl.Environ.Microbiol.,1999,65(1):278-282.
[14] Tan Y,Xu D,Li Y ,et al.Construction of a novel sacB -based system for marker-free gene deletion in Corynebacterium glutamicum[J].Plasmid,2012,67(1):44-52.
[15] Chen K C ,Ravichan dran A ,Guerrero A ,et al.The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin[J].Applied and Environmental Microbiology,2013,79(9):2899-2905.
[16] 钱栋宇.产酶溶杆菌OH11菌株几丁质酶基因的功能研究[D].南京:南京农业大学,2011.
[17] Puopolo G ,Cimmino A ,Palmieri M C ,et al.Lysobacter capsici AZ78 produces cyclo(L-Pro- L-Tyr),a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola[J].Journal of Applied Microbiology,2014,117(4):1168-1180.
[18] Xie Y,Wright S,Shen Y,et al.Bioactive natural products from Lysobacter[J].Nat.Prod.Rep.,2012,29(11):1277-1287.
[19] Hayward A C,Fegan N,Fegan M,et al.Stenotrophomonas and Lysobacter:ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology[J].Journal of Applied Microbiology,2009,108(3):756-770.
[20] Qian G,Hu B,Jiang Y,et al.Identification and characterization of Lysobacter enzymogenes as a biological control agent against some fungal pathogens[J].Agricultural Sciences in China,2009,8(1):68-75.
[21] 牟蕾,衣静莉,李星志,等.一株高拮抗活性菌株的鉴定及其抗菌活性缺失突变体的获得[J].山东农业科学,2018,50(7):126-132.
[22] 信欣,陈丽杰,薛闯.CRISPR-Cas9技术在细菌中的研究进展及应用[J].微生物学杂志,2018,38(6):97-102.
[23] Quandt J,Hynes M F.Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria[J].Gene,1993,127(1):15-21.
[24] 刘琳琳,钱国良,刘凤权.胶状溶杆菌OH17菌株遗传操作系统的构建[J].南京农业大学学报,2017,40(4):649-654.
[25] 钱国良.产酶溶杆菌生防相关基因的克隆、功能分析及工程菌构建[D].南京:南京农业大学,2009.
[2] Deng Q,Xiao L,Liu Y,et al.Streptomyces avermitilis industrial strain as cell factory for Ivermectin B1a production[J].Synthetic and Systems Biotechnology,2019,4(1):34-39.
[3] Fu Y,Yu Z,Liu S,et al.c-di-GMP regulates various phenotypes and insecticidal activity of Gram-positive Bacillus thuringiensis[J].Frontiers in Microbiology,2018,9:45.
[4] 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.
[5] El-Tarabily K A,Nassar A H,Hardy G E,et al.Plant growth promotion and biological control of Pythium aphanidermatum,a pathogen of cucumber,by endophytic actinomycetes[J].Journal of Applied Microbiology,2009,106(1):13-26.
[6] Zúňiga A,Poupin M J,Donoso R et al.Quorum sensing and indole-3-acetic acid degradation play a role in colonization and plant growth promotion of Arabidopsis thaliana by Burkholderia phytofirmans PsJN[J].Mol.Plant-Microbe Interact.,2013,26(5):546-553.
[7] 刘朝霞.辣椒溶杆菌X2-3抗菌物质的初步分离及其特性分析[D].泰安:山东农业大学,2016.
[8] Yi J L,Wang J,Li Q,et al.Draft genome sequence of the bacterium Lysobacter capsici X2-3,with a broad spectrum of antimicrobial activity against multiple plant-pathogenic microbes[J].Genome announcements,2015,3 (3):e00589-15.
[9] Qian G,Wang Y,Qian D,et al.Selection of available suicide vectors for gene mutagenesis using chiA (a chitinase encoding gene) as a new reporter and primary functional analysis of chiA in Lysobacter enzymogenes strain OH11[J].World J.Microbiol.Biotechnol.,2012,28(2):549-557.
[10] Qian G,Xu F,Venturi V,et al.Roles of a solo LuxR in the biological control agent Lysobacter enzymogenes strain OH11[J].Phytopathology,2014,104(3):224-231.
[11] Zou L F ,Li Yu R,Chen G Y .A non-marker mutagenesis strategy to generate poly-hrp gene mutants in the rice pathogen Xanthomonas oryzae pv.oryzicola[J].Agricultural Sciences in China,2011,10(8):1139-1150.
[12] Prentki P,Karch F,Iida S,et al.The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication[J].Gene,1981,14(4):289-299.
[13] Katzen F,Becker A,Verónica Ielmini M,et al.New mobilizable vectors suitable for gene replacement in gram-negative bacteria and their use in mapping of the 3' end of the Xanthomonas campestris pv.campestris gum operon[J].Appl.Environ.Microbiol.,1999,65(1):278-282.
[14] Tan Y,Xu D,Li Y ,et al.Construction of a novel sacB -based system for marker-free gene deletion in Corynebacterium glutamicum[J].Plasmid,2012,67(1):44-52.
[15] Chen K C ,Ravichan dran A ,Guerrero A ,et al.The Burkholderia contaminans MS14 ocfC gene encodes a xylosyltransferase for production of the antifungal occidiofungin[J].Applied and Environmental Microbiology,2013,79(9):2899-2905.
[16] 钱栋宇.产酶溶杆菌OH11菌株几丁质酶基因的功能研究[D].南京:南京农业大学,2011.
[17] Puopolo G ,Cimmino A ,Palmieri M C ,et al.Lysobacter capsici AZ78 produces cyclo(L-Pro- L-Tyr),a 2,5-diketopiperazine with toxic activity against sporangia of Phytophthora infestans and Plasmopara viticola[J].Journal of Applied Microbiology,2014,117(4):1168-1180.
[18] Xie Y,Wright S,Shen Y,et al.Bioactive natural products from Lysobacter[J].Nat.Prod.Rep.,2012,29(11):1277-1287.
[19] Hayward A C,Fegan N,Fegan M,et al.Stenotrophomonas and Lysobacter:ubiquitous plant-associated gamma-proteobacteria of developing significance in applied microbiology[J].Journal of Applied Microbiology,2009,108(3):756-770.
[20] Qian G,Hu B,Jiang Y,et al.Identification and characterization of Lysobacter enzymogenes as a biological control agent against some fungal pathogens[J].Agricultural Sciences in China,2009,8(1):68-75.
[21] 牟蕾,衣静莉,李星志,等.一株高拮抗活性菌株的鉴定及其抗菌活性缺失突变体的获得[J].山东农业科学,2018,50(7):126-132.
[22] 信欣,陈丽杰,薛闯.CRISPR-Cas9技术在细菌中的研究进展及应用[J].微生物学杂志,2018,38(6):97-102.
[23] Quandt J,Hynes M F.Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria[J].Gene,1993,127(1):15-21.
[24] 刘琳琳,钱国良,刘凤权.胶状溶杆菌OH17菌株遗传操作系统的构建[J].南京农业大学学报,2017,40(4):649-654.
[25] 钱国良.产酶溶杆菌生防相关基因的克隆、功能分析及工程菌构建[D].南京:南京农业大学,2009.