论文摘要
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明显提高30%以上。进一步对A1501菌中参与氧感应调控的Fnr-型转录因子Anr的功能进行了研究,结果表明anr基因的表达特异响应外界氧信号,其突变导致固氮酶活下降80%以上,生物膜形成能力下降25%左右,固氮及生物膜相关基因的表达显著下调。2.分别构建了gacS和gacA基因的缺失突变株并测定了基因缺失对菌体生长、固氮及生物膜形成能力的影响。结果显示,gacS和gacA基因的突变并不影响菌体生长,但gacA基因突变株固氮酶活相比野生型下降40%以上,gacS突变株酶活下降30%以上,实时定量分析表明,gacA基因突变株中nif基因（nifA,nifH,nifD,nifK）的表达量下调明显,而gacS突变株中nif基因的表达量上调。gacA突变株生物膜形成量明显减少（高达69%）,而gacS突变株的生物膜形成量则无明显变化。此外,两个基因突变均导致编码生物膜合成相关的小RNA（rsmX1,rsmX2,rsmY,rsmZ）及参与一般胁迫调控因子RpoS的基因表达显著下调。3.转录组分析了A1501野生型与gacA突变株在对数期和稳定期的表达谱差异。gacA突变导致对数期64个基因表达显著下调,29个基因表达上调;稳定期246个基因表达下调,211个基因表达上调。这些基因的功能可能与碳氮代谢、生物膜形成、运动及趋化、物质转运、逆境胁迫等相关。启动子分析表明,42基因的启动子区域含有保守的GacA识别序列,暗示GacA可能直接调控这些基因的转录表达。综上所述,施氏假单胞菌A1501中gacS和gacA基因可响应外界氧、温度等环境信号,参与固氮及生物膜形成相关基因的表达调控。本研究为全面了解固氮细菌的生物膜分子调控与生物固氮的协同调控及环境适应机制奠定了重要的理论基础。
论文目录
摘要ABSTRACTAbbreviationsChapter1 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 planChapter2 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 ConclusionChapter3 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 ConclusionChapter4 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 ConclusionChapter5 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 conditionReferenceAppendixACKNOWLEDGEMENTRESUME Personal Profile Academic Qualification Award and Fellowship Research Publication Thesis titled
文章来源
类型: 博士论文
作者: Muhammad Ali Rasheed
导师: 林敏
关键词: 施氏假单胞菌,双组分调控系统,生物固氮,生物膜形成,转录组分析
来源: 中国农业科学院
年度: 2019
分类: 基础科学
专业: 生物学,生物学
单位: 中国农业科学院
分类号: Q933
总页数: 141
文件大小: 6945K
下载量: 224
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标签：施氏假单胞菌论文; 双组分调控系统论文; 生物固氮论文; 生物膜形成论文; 转录组分析论文;
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
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