施氏假单胞菌A1501双组分调节系统GacS/GacA参与固氮及生物膜形成的功能解析
发布时间：2021-01-24 05:16
双组份调控系统gacS-gacA广泛存在于革兰氏阴性细菌中,其中gacS编码一个信号感应激酶,gacA编码转录调控因子。关于GacS-GacA功能的研究主要集中在肠道细菌和假单胞菌中。以模式菌铜绿假单胞菌为例,gacS-gacA系统作为全局调节因子参与调控次生代谢产物合成、生物膜形成和生态适应性等生理过程。施氏假单胞菌A1501分离自水稻根际,是目前报道的少数几株具有固氮能力的假单胞菌,该菌能在水稻根部定殖形成生物膜并固氮生长。基因组分析表明,该菌中具有一套编码GacS-GacA系统的基因,但其是否参与生物膜形成和固氮调控尚不明确。本研究中,我们对gacS-gacA基因的表达特性、在生物膜形成及生物固氮过程中的功能进行了研究,取得以下研究结果:1.分析了不同非生物胁迫及生物膜形成条件下施氏假单胞菌A1501中gacS和gacA基因的表达规律。结果表明,gacS和gacA基因的表达水平受外界温度、氮源、盐浓度、氧浓度等环境信号不同程度的影响,在生物膜形成能力强的环境下gacS和gacA基因的表达高水平诱导。此外,gacA基因在微好氧条件（氧气浓度0.5%）高表达,比正常培养条件下的表达量...
【文章来源】：中国农业科学院北京市
【文章页数】：141 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Abbreviations
Chapter1 Introduction
1.1 GacS/GacA,two component system
1.2 Pseudomonas stutzeri A1501
1.3 Signaling pathway and its role with GacS/GacA two component system in Pseudomonas
1.4 Native and synthetic gene regulation to nitrogen limitation stress
1.5 Role of sigma factors and its importance in stress
1.6 Research plan
Chapter2 Demonstrating gacS and gacA activity during different abiotic conditions in Pseudomonas stutzeri A1501
2.1 Introduction
2.2 Methods and material
2.2.1 Bacterial strains,culture media and growth condition:
2.2.2 Analysis at abiotic stress
2.2.3 Nitrogenase activity assays
2.2.4 Quantitative real-time qRT-PCR analysis
2.3 Results
2.3.1 Effect of high osmolarty
2.3.2 Effect of nitrogen starvation on biofilm formation
2.3.3 Effect of low pH
2.3.4 Effect of extreme temperature
2.4 Biofilm formation at different oxygen levels
2.5 Nitrogenase activity different oxygen concentrations
2.6 Conclusion
Chapter3 The effect of mutation of anr gene on nitrogen fixation and biofilm formation in Pseudomonas stutzeri A1501 at low levels of oxygen
3.1 Introduction
3.2 Method and Materials
3.2.1 Bacterial strains,culture media,plasmids and growth condition
3.2.2 Growth curve analysis
3.2.3 Oxidative stress analysis:
3.2.4 Estimation of biofilm formation
3.2.5 Nitrogenase activity assays
3.2.6 Quantitative Real-Time PCR analysis
3.3 Results
3.4 Construction of anr insertional mutant
Amplification for anr region of gene for insertion mutation
3.5 pJET assembly of gene:
3.6 JET plasmid transformed in E.coli:
3.7 Formation of pK-18 mob with anr plasmid and transformation in E.coli
3.8 Colony PCR
3.9 Tri-parental mating or Conjugation:
3.9.1 Pre-cultures
3.9.2 Conjugation
3.10 Construction of dnr insertional mutant
3.10.1 Amplification for dnr region of gene for insertion mutation
3.10.2 Ligating the plasmid pK-18 mob with dnr fragment
3.11 Tri-parental mating or conjugation:
3.11.1 Pre-cultures
3.11.2 Conjugation
3.12 Complimentary Mutant formation:
3.12.1 Cutting the Plasmid using restriction enzymes:
3.12.2 Ligating the plasmid pLAFR-3 with anr and dnr gene
3.12.3 Escherichia coli transformation:
3.12.4 Colony PCR
3.12.5 Pre-cultures
3.12.6 Conjugation
3.13 Bioinformatics Analysis
3.13.1 Growth curve analysis
3.13.2 Effect of different oxygen concentration on gacS,gacA and anr
3.14 Oxidative stress analysis:
3.15 Studying biofilm dispersal of anr and dnr genes
Test tube Biofilm formation
Relative qRT-PCR analysis for biofilm formation
3.16 Nitrogenase activity anr and dnr genes
3.17 Relative qRT-PCR analysis for nitrogenase activity
3.18 Conclusion
Chapter4 Functional and regulatory characterization of GacS/GacA two component system in plant associated microorganism Pseudomonas stutzeri A1501
4.1 Introduction
4.2 Materials and Method
4.2.1 Bacterial strains,culture media,plasmids and growth condition
4.2.2 Strains and plasmids
4.2.3 Medium
4.2.4 Enzymes and chemical reagents
4.2.5 Major instruments
4.2.6 Commonly used solution and antibiotics
4.3 Experimental methods
Extraction of bacterial plasmid DNA
4.3.1 Isolation of Pseudomonas stutzeri A1501 genome:
4.3.2 PCR amplification of genes
4.4 Colony PCR
4.5 Extraction of DNA from gel
4.6 Tri-parental mating or conjugation
4.6.1 Pre-cultures
4.6.2 Conjugation
4.7 Selection of the first recombination event
4.8 Selection of the second recombination event
4.9 Complimentary mutant formation
4.9.0 Amplification of the gene of interest
4.9.1 Cutting the plasmid using restriction enzymes
4.9.2 Ligating the plasmid pLAFR-3 with gacS and gacA gene
4.9.3 Escherichia coli transformation
4.9.4 Colony PCR
4.9.5 Pre-cultures
4.9.6 Conjugation
4.10 Construction of double mutant（ΔgacS/ ΔgacA mutant）
4.11 Extraction Of bacterial total RNA
4.12 cDNA reverse transcription synthesis
4.13 Growth curve analysis
4.13.1 Analysis at abiotic stress using96 well plate for estimation of biofilm formation
4.13.2 Nitrogenase activity assays
4.13.3 Quantitative real-time qRT-PCR analysis
4.14 Fluorescence real-time quantitative PCR
4.15 Probe design principles
4.16 Results
4.16.1 Ligating the up and down regulation gene of gacS with gmR（gentamycin resistant gene）
4.16.2 Ligating the plasmid pK18 mob-sacB with up and down regulatory gene of gacS with gmR（gentamycin resistant gene）
4.17 Tri-parental mating or conjugation
4.17.1 After first cross
4.17.2 After second cross
4.18 Complimentary mutant formation:
Amplification of the gene of interest
Transformation of pLAFR-3 along with gacS gene in E.coli
4.19 Construction of double mutant（ΔgacS/ ΔgacA mutant）
Relative qRT-PCR analysis for nitrogenase activity
4.20 Bioinformatics Analysis:
4.21 Growth curve analysis
4.22 Effect of different abiotic stress on biofilm formation
4.23 Effect of nitrogen starvation condition
4.24 Effect of low pH
4.25 Effect of temperature
4.26 Nitrogenase activity
4.27 Biofilm formation
4.28 Conclusion
Chapter5 Transcriptome analysis of gacA mutant in Pseudomonas stutzeri A1501 during normal growth curve
5.1 Introduction
5.2 Method and Materials:
5.2.1 Bacterial strains,culture media,plasmids and growth condition
5.2.2 Growth curve analysis
5.2.3 Sample collection
5.2.4 RNA isolation
5.2.5 RNA deep seq data analysis
5.3 Results
5.3.1 Influence of gacA inactivation on transcriptome profile
5.3.2 gacA targets involved in primary metabolism and energy metabolism
5.3.3 Total number of sRNA:
5.4 Conclusion:
Discussion
Role of sRNA in stress condition
Reference
Appendix
ACKNOWLEDGEMENT
RESUME
Personal Profile
Academic Qualification
Award and Fellowship
Research Publication
Thesis titled
本文编号：2996646
【文章来源】：中国农业科学院北京市
【文章页数】：141 页
【学位级别】：博士
【文章目录】：
摘要
ABSTRACT
Abbreviations
Chapter1 Introduction
1.1 GacS/GacA,two component system
1.2 Pseudomonas stutzeri A1501
1.3 Signaling pathway and its role with GacS/GacA two component system in Pseudomonas
1.4 Native and synthetic gene regulation to nitrogen limitation stress
1.5 Role of sigma factors and its importance in stress
1.6 Research plan
Chapter2 Demonstrating gacS and gacA activity during different abiotic conditions in Pseudomonas stutzeri A1501
2.1 Introduction
2.2 Methods and material
2.2.1 Bacterial strains,culture media and growth condition:
2.2.2 Analysis at abiotic stress
2.2.3 Nitrogenase activity assays
2.2.4 Quantitative real-time qRT-PCR analysis
2.3 Results
2.3.1 Effect of high osmolarty
2.3.2 Effect of nitrogen starvation on biofilm formation
2.3.3 Effect of low pH
2.3.4 Effect of extreme temperature
2.4 Biofilm formation at different oxygen levels
2.5 Nitrogenase activity different oxygen concentrations
2.6 Conclusion
Chapter3 The effect of mutation of anr gene on nitrogen fixation and biofilm formation in Pseudomonas stutzeri A1501 at low levels of oxygen
3.1 Introduction
3.2 Method and Materials
3.2.1 Bacterial strains,culture media,plasmids and growth condition
3.2.2 Growth curve analysis
3.2.3 Oxidative stress analysis:
3.2.4 Estimation of biofilm formation
3.2.5 Nitrogenase activity assays
3.2.6 Quantitative Real-Time PCR analysis
3.3 Results
3.4 Construction of anr insertional mutant
Amplification for anr region of gene for insertion mutation
3.5 pJET assembly of gene:
3.6 JET plasmid transformed in E.coli:
3.7 Formation of pK-18 mob with anr plasmid and transformation in E.coli
3.8 Colony PCR
3.9 Tri-parental mating or Conjugation:
3.9.1 Pre-cultures
3.9.2 Conjugation
3.10 Construction of dnr insertional mutant
3.10.1 Amplification for dnr region of gene for insertion mutation
3.10.2 Ligating the plasmid pK-18 mob with dnr fragment
3.11 Tri-parental mating or conjugation:
3.11.1 Pre-cultures
3.11.2 Conjugation
3.12 Complimentary Mutant formation:
3.12.1 Cutting the Plasmid using restriction enzymes:
3.12.2 Ligating the plasmid pLAFR-3 with anr and dnr gene
3.12.3 Escherichia coli transformation:
3.12.4 Colony PCR
3.12.5 Pre-cultures
3.12.6 Conjugation
3.13 Bioinformatics Analysis
3.13.1 Growth curve analysis
3.13.2 Effect of different oxygen concentration on gacS,gacA and anr
3.14 Oxidative stress analysis:
3.15 Studying biofilm dispersal of anr and dnr genes
Test tube Biofilm formation
Relative qRT-PCR analysis for biofilm formation
3.16 Nitrogenase activity anr and dnr genes
3.17 Relative qRT-PCR analysis for nitrogenase activity
3.18 Conclusion
Chapter4 Functional and regulatory characterization of GacS/GacA two component system in plant associated microorganism Pseudomonas stutzeri A1501
4.1 Introduction
4.2 Materials and Method
4.2.1 Bacterial strains,culture media,plasmids and growth condition
4.2.2 Strains and plasmids
4.2.3 Medium
4.2.4 Enzymes and chemical reagents
4.2.5 Major instruments
4.2.6 Commonly used solution and antibiotics
4.3 Experimental methods
Extraction of bacterial plasmid DNA
4.3.1 Isolation of Pseudomonas stutzeri A1501 genome:
4.3.2 PCR amplification of genes
4.4 Colony PCR
4.5 Extraction of DNA from gel
4.6 Tri-parental mating or conjugation
4.6.1 Pre-cultures
4.6.2 Conjugation
4.7 Selection of the first recombination event
4.8 Selection of the second recombination event
4.9 Complimentary mutant formation
4.9.0 Amplification of the gene of interest
4.9.1 Cutting the plasmid using restriction enzymes
4.9.2 Ligating the plasmid pLAFR-3 with gacS and gacA gene
4.9.3 Escherichia coli transformation
4.9.4 Colony PCR
4.9.5 Pre-cultures
4.9.6 Conjugation
4.10 Construction of double mutant（ΔgacS/ ΔgacA mutant）
4.11 Extraction Of bacterial total RNA
4.12 cDNA reverse transcription synthesis
4.13 Growth curve analysis
4.13.1 Analysis at abiotic stress using96 well plate for estimation of biofilm formation
4.13.2 Nitrogenase activity assays
4.13.3 Quantitative real-time qRT-PCR analysis
4.14 Fluorescence real-time quantitative PCR
4.15 Probe design principles
4.16 Results
4.16.1 Ligating the up and down regulation gene of gacS with gmR（gentamycin resistant gene）
4.16.2 Ligating the plasmid pK18 mob-sacB with up and down regulatory gene of gacS with gmR（gentamycin resistant gene）
4.17 Tri-parental mating or conjugation
4.17.1 After first cross
4.17.2 After second cross
4.18 Complimentary mutant formation:
Amplification of the gene of interest
Transformation of pLAFR-3 along with gacS gene in E.coli
4.19 Construction of double mutant（ΔgacS/ ΔgacA mutant）
Relative qRT-PCR analysis for nitrogenase activity
4.20 Bioinformatics Analysis:
4.21 Growth curve analysis
4.22 Effect of different abiotic stress on biofilm formation
4.23 Effect of nitrogen starvation condition
4.24 Effect of low pH
4.25 Effect of temperature
4.26 Nitrogenase activity
4.27 Biofilm formation
4.28 Conclusion
Chapter5 Transcriptome analysis of gacA mutant in Pseudomonas stutzeri A1501 during normal growth curve
5.1 Introduction
5.2 Method and Materials:
5.2.1 Bacterial strains,culture media,plasmids and growth condition
5.2.2 Growth curve analysis
5.2.3 Sample collection
5.2.4 RNA isolation
5.2.5 RNA deep seq data analysis
5.3 Results
5.3.1 Influence of gacA inactivation on transcriptome profile
5.3.2 gacA targets involved in primary metabolism and energy metabolism
5.3.3 Total number of sRNA:
5.4 Conclusion:
Discussion
Role of sRNA in stress condition
Reference
Appendix
ACKNOWLEDGEMENT
RESUME
Personal Profile
Academic Qualification
Award and Fellowship
Research Publication
Thesis titled
本文编号：2996646
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