| abstract | 第9-11页 |
| 摘要 | 第12-14页 |
| LIST OF ABBREVIATIONS | 第14-16页 |
| 1. INTRODUCTION | 第16-43页 |
| 1.1. Epidemiology of Brucellosis | 第16-19页 |
| 1.2. Brucella pathogenesis | 第19-21页 |
| 1.3. Brucella modulation of innate immune response | 第21-24页 |
| 1.4. Brucella impressive mechanisms to evade adaptive immune response | 第24-27页 |
| 1.5. Putative role of small non-coding RNAs against Brucella stealth strategy | 第27-28页 |
| 1.6. Mechanisms employed by bacterial sRNAs for gene regulation | 第28-30页 |
| 1.7. Recent approaches for identification of novel small non-coding RNAs | 第30-36页 |
| 1.7.1. Labeling of total RNA | 第31页 |
| 1.7.2. Identification of functional sRNA by genetic screens | 第31-32页 |
| 1.7.3. Identification of sRNA based on computational predictions | 第32页 |
| 1.7.4. RNomics and RNA-Seq (next-generation sequencing) | 第32-33页 |
| 1.7.5. Identification of sRNA based on DNA microarray analysis | 第33-34页 |
| 1.7.6. Co-purification of sRNAs with proteins | 第34-35页 |
| 1.7.7. Genomic SELEX | 第35页 |
| 1.7.8. Prediction of sRNAs based on comparative genomics | 第35-36页 |
| 1.8. Approaches to identify targets of non-coding RNAs | 第36-40页 |
| 1.8.1. Genetic approaches | 第36-37页 |
| 1.8.2. Bioinformatics-aided approaches | 第37-39页 |
| 1.8.3. Proteomics approaches | 第39页 |
| 1.8.4. Transcriptomic approaches | 第39-40页 |
| 1.9. Target validation approaches | 第40-41页 |
| 1.9.1. In vivo approaches | 第40-41页 |
| 1.9.2. In vitro approaches | 第41页 |
| 1.10. Study background | 第41-42页 |
| 1.11. Objective of this study | 第42-43页 |
| 2. MATERIALS AND METHODS | 第43-69页 |
| 2.1. Materials | 第43-53页 |
| 2.1.1. Labware and instruments | 第43-44页 |
| 2.1.2. Tool kits,enzymes and antibodies | 第44-45页 |
| 2.1.3. Preparation of media and reagents | 第45-53页 |
| 2.2. Bacterial strains,plasmids and media | 第53-54页 |
| 2.3. Total RNA isolation | 第54页 |
| 2.4. 5' RACE | 第54-56页 |
| 2.5. Construction of target and AbcR1 plasmids | 第56-57页 |
| 2.6. Construction of AbcR1 and target mRNA mutants | 第57页 |
| 2.7. Construction of shuttle plasmid | 第57-58页 |
| 2.8. Determination of copy number of shuttle plasmid | 第58页 |
| 2.9. Purification of PCR product using gel extraction Kit | 第58-59页 |
| 2.10. Purification of PCR product using ethanol | 第59页 |
| 2.11. Preparation of B. melitensis 16M competent cells | 第59-60页 |
| 2.12. Extraction of low-copy plasmid on large scale | 第60-61页 |
| 2.13. Ligation of target fragments with vector pXG-10SF | 第61-62页 |
| 2.14. Transformation of enzyme-linked product or recombinant product | 第62页 |
| 2.15. Plasmid extraction using Plasmid Miniprep (OMEGA kit) | 第62-63页 |
| 2.16. Determination of fluorescence on agar plates | 第63页 |
| 2.17. Fluorescence measurement of liquid cultures | 第63-64页 |
| 2.18. Electroporation of plasmids into Brucella | 第64页 |
| 2.19. Detection of GFP protein content with Western blot | 第64-66页 |
| 2.20. Detection of target mRNAs expression by qRT-PCR | 第66-68页 |
| 2.21. Bio-computational predictions | 第68页 |
| 2.22. Statistical analysis | 第68-69页 |
| 3. RESULTS AND ANALYSIS | 第69-94页 |
| 3.1. sRNA AbcR1 structure prediction | 第69-70页 |
| 3.2. sRNA AbcR1 conservative analysis in Brucella | 第70-71页 |
| 3.3. Computational predictions of AbcR1 sRNA targets in B. melitensis | 第71-74页 |
| 3.4. PCR amplification of target mRNAs | 第74-75页 |
| 3.5. Construction of plasmids used for co-transformation | 第75-79页 |
| 3.5.1. pXG-0 | 第75页 |
| 3.5.2. pXG-10SF and construction of target mRNA plasmids | 第75-76页 |
| 3.5.3. pJV300 and construction of sRNA AbcR1 plasmid | 第76-78页 |
| 3.5.4. General strategy of plasmid fusion | 第78-79页 |
| 3.6. Experimental validation and regulation of sRNA AbcR1 expression in B. melitensis targets | 第79-84页 |
| 3.7. The conserved seed region in sRNA AbcR1 regulates multiple targets of B. melitensis | 第84-89页 |
| 3.8. Construction of novel shuttle expression vector | 第89-90页 |
| 3.9. Determination of copy number of shuttle plasmid | 第90-92页 |
| 3.10. Validation of sRNA AbcR1 regulation in B. melitensis 16M competent cells | 第92-94页 |
| 4. DISCUSSION | 第94-99页 |
| 4.1. Contribution of sRNAs in Brucella stealthy behaviour | 第94-96页 |
| 4.2. Prediction of sRNA AbcR1 targets in B. melitensis | 第96页 |
| 4.3. Experimental validation of sRNA AbcR1-mRNA regulation in B. melitensis | 第96-97页 |
| 4.4. Conserved seed region of sRNA AbcR1 plays essential role in target mRNA regulation | 第97页 |
| 4.5. Determination of sRNA AbcR1-mRNA regulation in B. melitensis competent cells | 第97-99页 |
| 5. CONCLUSION | 第99-100页 |
| REFERENCES | 第100-121页 |
| PUBLICATIONS | 第121-122页 |
| ACKNOWLEDGEMENT | 第122-123页 |
