植物根际促生菌Bacillus mycoides Gnyt1铁载体分泌相关功能基因的挖掘
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摘要
【目的】为挖掘优良促生菌(PGPR)菌株,分析和定位功能基因,发现其科学价值和工业化开发,为农业生产服务。【方法】以Bacillus mycoides Gnyt1菌株为材料,采用二代和三代测序技术相结合的研究体系,对菌株进行全基因组测序研究,分析菌株核基因组可能存在的功能基因和菌株分泌铁载体相关的基因。【结果】本研究基因组序列拼接后总长度为5597907 bp,GC百分含量为35.57%,该菌株中与铁载体分泌相关的基因共9条,其中2条基因主要存在于Porphyrin metabolism途径,与细胞内铁的运输代谢有关。【结论】通过全基因组测序,最终确定了与铁载体分泌相关的功能基因为GYT1和GYT2,为下一步基因功能验证奠定了基础。
[Objective] For the excavation of plant growth promoting rhizobacteria(PGPR) strains, the functional genes were analyzed and mapped in order to find out their scientific value and industrialization development and serve agricultural production. [Methods] Bacillus mycoides Gnyt1 strain was used as the material, and the second and third generation sequencing technology were combined to carry out the whole genome sequencing of the strain.The possible functional genes of the nuclear genome of the strain were analyzed, and genes secreting iron carrier-related strain were analyzed. [Results] The total length of the genomic sequence after splicing was 5597907 bp.The percentage of GC was 35.57%. There were 9 genes involved in the secretion of siderophore in this strain, of which 2 genes mainly existed in the Porphyrin metabolism pathway and were associated with intracellular iron transport metabolism related. [Conclusion] Through whole genome sequencing, the functional genes related to the secretion of siderophores were identified as GYT1 and GYT2, which makes it possible for verifying the gene functions.
[Objective] For the excavation of plant growth promoting rhizobacteria(PGPR) strains, the functional genes were analyzed and mapped in order to find out their scientific value and industrialization development and serve agricultural production. [Methods] Bacillus mycoides Gnyt1 strain was used as the material, and the second and third generation sequencing technology were combined to carry out the whole genome sequencing of the strain.The possible functional genes of the nuclear genome of the strain were analyzed, and genes secreting iron carrier-related strain were analyzed. [Results] The total length of the genomic sequence after splicing was 5597907 bp.The percentage of GC was 35.57%. There were 9 genes involved in the secretion of siderophore in this strain, of which 2 genes mainly existed in the Porphyrin metabolism pathway and were associated with intracellular iron transport metabolism related. [Conclusion] Through whole genome sequencing, the functional genes related to the secretion of siderophores were identified as GYT1 and GYT2, which makes it possible for verifying the gene functions.
引文
[1]Sun GZ,Yao T,Feng CJ,Chen L,Li JH,Wang LD.Identification and biocontrol potential of antagonistic bacteria strains against Sclerotinia sclerotiorum and their growth-promoting effects on Brassica napus.Biological Control,2017,104:35-43.
[2]门晋名.铁载体高产菌的筛选及铁载体的分离纯化.青岛科技大学硕士学位论文,2013.
[3]Long YC,Chen X,Zhou SQ.Isolation,identification and assessment on selenium biofortification of siderophore-producing rhizobacteria.Current Biotechnology,2017,7(5):402-408.(in Chinese)龙云川,陈轩,周少奇.高产铁载体根际菌的筛选鉴定及硒活化特性评价.生物技术进展,2017,7(5):402-408.
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[5]Loaces I,Ferrando L,Scavino AF.Dynamics,diversity and function of endophytic siderophore-producing bacteria in rice.Microbial Ecology,2011,61(3):606-618.
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[7]Li JH.The characteristics of a fine plant growth-promoting rhizobacteria Bacillus mycoides Gnyt1 and its whole genome sequencing analysis.Doctor Dissertation of Gansu Agricultural University,2017.(in Chinese)李建宏.优良植物根际促生菌Bacillus mycoides Gnyt1特性研究及全基因组测序分析.甘肃农业大学博士学位论文,2017.
[8]Zhao X,Xie ZX,Chen SX,Shen P.Improvement and analysis in over-siderophores production bacteria filtrating and detecting.Microbiology China,2006,33(6):95-98.(in Chinese)赵翔,谢志雄,陈绍兴,沈萍.适合高产铁载体细菌筛选、检测体系的改进与探析.微生物学通报,2006,33(6):95-98.
[9]Green MR,Sambrook J.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor,NY:Cold Spring Harbor Laboratory Press,2012.
[10]Wendl MC.Occupancy modeling of coverage distribution for whole genome shotgun DNA sequencing.Bulletin of Mathematical Biology,2006,68(1):179-196.
[11]Hendre PS,Kamalakannan R,Rajkumar R,Varghese M.High-throughput targeted SNP discovery using Next Generation Sequencing(NGS)in few selected candidate genes in Eucalyptus camaldulensis.BMC Proceedings,2011,5(S7):O17.
[12]Frey KG,Herrera-Galeano JE,Redden CL,Luu TV,Servetas SL,Mateczun AJ,Mokashi VP,Bishop-Lilly KA.Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood.BMC Genomics,2014,15(1):96.
[13]Moriya Y,Itoh M,Okuda S,Yoshizawa AC,Kanehisa M.KAAS:an automatic genome annotation and pathway reconstruction server.Nucleic Acids Research,2007,35(S2):W182-W185.
[14]Dong H,Rui JP,Sun JN,Li XZ,Mao XZ.Complete genome sequencing and diversity analysis of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.Acta Microbiologica Sinica,2017,57(11):1716-1721.(in Chinese)董浩,芮俊鹏,孙建安,李香真,毛相朝.嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析.微生物学报,2017,57(11):1716-1721.
[15]Cui YH,Dong AJ,Qu XJ.Siderophores-mediated iron uptake system of microorganisms.Chemistry of Life,2008,28(6):786-790.(in Chinese)崔艳华,董爱军,曲晓军.微生物铁载体运输系统.生命的化学,2008,28(6):786-790.
[16]Ji C,Miller MJ.Chemical syntheses and in vitro antibacterial activity of two desferrioxamine B-ciprofloxacin conjugates with potential esterase and phosphatase triggered drug release linkers.Bioorganic&Medicinal Chemistry,2012,20(12):3828-3836.
[17]Mislin GLA,Schalk IJ.Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.Metallomics,2014,6(3):408-420.
[18]M?llmann U,Heinisch L,Bauernfeind A,K?hler T,Ankel-Fuchs D.Siderophores as drug delivery agents:application of the“Trojan Horse”strategy.Biometals,2009,22(4):615-624.
[19]Page MGP.Siderophore conjugates.Annals of the New York Academy of Sciences,2013,1277(1):115-126.
[20]Zheng TF,Nolan EM.Enterobactin-mediated delivery ofβ-lactam antibiotics enhances antibacterial activity against pathogenic Escherichia coli.Journal of the American Chemical Society,2014,136(27):9677-9691.
[21]Chu BC,Garcia-Herrero A,Johanson TH,Krewulak KD,Lau CK,Peacock RS,Slavinskaya Z,Vogel HJ.Siderophore uptake in bacteria and the battle for iron with the host;Abird’s eye view.Biometals,2010,23(4):601-611.
[22]Mor H,Barash I.Characterization of siderophore-mediated iron transport in Geotrichum candidum,A non-siderophore producer.Biology of Metals,1990,2(4):209-213.
[2]门晋名.铁载体高产菌的筛选及铁载体的分离纯化.青岛科技大学硕士学位论文,2013.
[3]Long YC,Chen X,Zhou SQ.Isolation,identification and assessment on selenium biofortification of siderophore-producing rhizobacteria.Current Biotechnology,2017,7(5):402-408.(in Chinese)龙云川,陈轩,周少奇.高产铁载体根际菌的筛选鉴定及硒活化特性评价.生物技术进展,2017,7(5):402-408.
[4]Li YM,Wang QY,Tu WG,Cui YL,Zhong QD,Li LH,Chen Q,Yu XM.Growth promoting activity of siderophore secreting bacteria for peanut plant under nickel stress.Microbiology China,2017,44(8):1882-1890.(in Chinese)李艳梅,王琼瑶,涂卫国,崔永亮,钟玘狄,李俐珩,陈强,余秀梅.镍胁迫下产铁载体细菌对花生的促生性.微生物学通报,2017,44(8):1882-1890.
[5]Loaces I,Ferrando L,Scavino AF.Dynamics,diversity and function of endophytic siderophore-producing bacteria in rice.Microbial Ecology,2011,61(3):606-618.
[6]Liu T,Yao T,Chen JG,Ma WB,Liu H,Ma CY,Jiang YM.Identification and study on the effects of plant growth promoting rhizobacteria of Carex enervis.Acta Prataculturae Sinica,2016,25(12):130-139.(in Chinese)刘婷,姚拓,陈建纲,马文彬,刘欢,马骢毓,蒋永梅.无脉苔草根际优良促生菌鉴定及其作用研究.草业学报,2016,25(12):130-139.
[7]Li JH.The characteristics of a fine plant growth-promoting rhizobacteria Bacillus mycoides Gnyt1 and its whole genome sequencing analysis.Doctor Dissertation of Gansu Agricultural University,2017.(in Chinese)李建宏.优良植物根际促生菌Bacillus mycoides Gnyt1特性研究及全基因组测序分析.甘肃农业大学博士学位论文,2017.
[8]Zhao X,Xie ZX,Chen SX,Shen P.Improvement and analysis in over-siderophores production bacteria filtrating and detecting.Microbiology China,2006,33(6):95-98.(in Chinese)赵翔,谢志雄,陈绍兴,沈萍.适合高产铁载体细菌筛选、检测体系的改进与探析.微生物学通报,2006,33(6):95-98.
[9]Green MR,Sambrook J.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor,NY:Cold Spring Harbor Laboratory Press,2012.
[10]Wendl MC.Occupancy modeling of coverage distribution for whole genome shotgun DNA sequencing.Bulletin of Mathematical Biology,2006,68(1):179-196.
[11]Hendre PS,Kamalakannan R,Rajkumar R,Varghese M.High-throughput targeted SNP discovery using Next Generation Sequencing(NGS)in few selected candidate genes in Eucalyptus camaldulensis.BMC Proceedings,2011,5(S7):O17.
[12]Frey KG,Herrera-Galeano JE,Redden CL,Luu TV,Servetas SL,Mateczun AJ,Mokashi VP,Bishop-Lilly KA.Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood.BMC Genomics,2014,15(1):96.
[13]Moriya Y,Itoh M,Okuda S,Yoshizawa AC,Kanehisa M.KAAS:an automatic genome annotation and pathway reconstruction server.Nucleic Acids Research,2007,35(S2):W182-W185.
[14]Dong H,Rui JP,Sun JN,Li XZ,Mao XZ.Complete genome sequencing and diversity analysis of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.Acta Microbiologica Sinica,2017,57(11):1716-1721.(in Chinese)董浩,芮俊鹏,孙建安,李香真,毛相朝.嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析.微生物学报,2017,57(11):1716-1721.
[15]Cui YH,Dong AJ,Qu XJ.Siderophores-mediated iron uptake system of microorganisms.Chemistry of Life,2008,28(6):786-790.(in Chinese)崔艳华,董爱军,曲晓军.微生物铁载体运输系统.生命的化学,2008,28(6):786-790.
[16]Ji C,Miller MJ.Chemical syntheses and in vitro antibacterial activity of two desferrioxamine B-ciprofloxacin conjugates with potential esterase and phosphatase triggered drug release linkers.Bioorganic&Medicinal Chemistry,2012,20(12):3828-3836.
[17]Mislin GLA,Schalk IJ.Siderophore-dependent iron uptake systems as gates for antibiotic Trojan horse strategies against Pseudomonas aeruginosa.Metallomics,2014,6(3):408-420.
[18]M?llmann U,Heinisch L,Bauernfeind A,K?hler T,Ankel-Fuchs D.Siderophores as drug delivery agents:application of the“Trojan Horse”strategy.Biometals,2009,22(4):615-624.
[19]Page MGP.Siderophore conjugates.Annals of the New York Academy of Sciences,2013,1277(1):115-126.
[20]Zheng TF,Nolan EM.Enterobactin-mediated delivery ofβ-lactam antibiotics enhances antibacterial activity against pathogenic Escherichia coli.Journal of the American Chemical Society,2014,136(27):9677-9691.
[21]Chu BC,Garcia-Herrero A,Johanson TH,Krewulak KD,Lau CK,Peacock RS,Slavinskaya Z,Vogel HJ.Siderophore uptake in bacteria and the battle for iron with the host;Abird’s eye view.Biometals,2010,23(4):601-611.
[22]Mor H,Barash I.Characterization of siderophore-mediated iron transport in Geotrichum candidum,A non-siderophore producer.Biology of Metals,1990,2(4):209-213.