施氏假單胞菌A1501雙組分調(diào)節(jié)系統(tǒng)GacS/GacA參與固氮及生物膜形成的功能解析
發(fā)布時(shí)間：2021-01-24 05:16
雙組份調(diào)控系統(tǒng)gacS-gacA廣泛存在于革蘭氏陰性細(xì)菌中,其中g(shù)acS編碼一個(gè)信號(hào)感應(yīng)激酶,gacA編碼轉(zhuǎn)錄調(diào)控因子。關(guān)于GacS-GacA功能的研究主要集中在腸道細(xì)菌和假單胞菌中。以模式菌銅綠假單胞菌為例,gacS-gacA系統(tǒng)作為全局調(diào)節(jié)因子參與調(diào)控次生代謝產(chǎn)物合成、生物膜形成和生態(tài)適應(yīng)性等生理過(guò)程。施氏假單胞菌A1501分離自水稻根際,是目前報(bào)道的少數(shù)幾株具有固氮能力的假單胞菌,該菌能在水稻根部定殖形成生物膜并固氮生長(zhǎng)�；蚪M分析表明,該菌中具有一套編碼GacS-GacA系統(tǒng)的基因,但其是否參與生物膜形成和固氮調(diào)控尚不明確。本研究中,我們對(duì)gacS-gacA基因的表達(dá)特性、在生物膜形成及生物固氮過(guò)程中的功能進(jìn)行了研究,取得以下研究結(jié)果:1.分析了不同非生物脅迫及生物膜形成條件下施氏假單胞菌A1501中g(shù)acS和gacA基因的表達(dá)規(guī)律。結(jié)果表明,gacS和gacA基因的表達(dá)水平受外界溫度、氮源、鹽濃度、氧濃度等環(huán)境信號(hào)不同程度的影響,在生物膜形成能力強(qiáng)的環(huán)境下gacS和gacA基因的表達(dá)高水平誘導(dǎo)。此外,gacA基因在微好氧條件（氧氣濃度0.5%）高表達(dá),比正常培養(yǎng)條件下的表達(dá)量...
【文章來(lái)源】：中國(guó)農(nóng)業(yè)科學(xué)院北京市
【文章頁(yè)數(shù)】：141 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：2996646
【文章來(lái)源】：中國(guó)農(nóng)業(yè)科學(xué)院北京市
【文章頁(yè)數(shù)】：141 頁(yè)
【學(xué)位級(jí)別】：博士
【文章目錄】：
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
本文編號(hào)：2996646
本文鏈接：http://sikaile.net/projectlw/swxlw/2996646.html
最近更新
教材專著
