| Abstract | 第4-5页 |
| 摘要 | 第6-12页 |
| 1 Introduction | 第12-24页 |
| 1.1 MicroRNAs:The Master regulator in gene regulation | 第12-17页 |
| 1.1.1 Small RNA biogenesis pathway | 第12-14页 |
| 1.1.2 Functions of miRNAs in plants | 第14-15页 |
| 1.1.3 Co-suppression and RNA-mediated Gene Silencing:A brief history | 第15-16页 |
| 1.1.4 Silencing and counter Silencing | 第16-17页 |
| 1.2 RNA-Seq:Revolution in transcriptomics | 第17-19页 |
| 1.2.1 Introduction | 第17-18页 |
| 1.2.2 Brief history of RNA-Seq development | 第18页 |
| 1.2.3 Application of high-throughput RNA sequencing in plants | 第18-19页 |
| 1.3 Grafting and distant-grafting in plants | 第19-20页 |
| 1.4 Translocation of signaling molecules in plants | 第20-21页 |
| 1.5 Distant-grafting between Lycium chinense Mill.(gouqi) and tomato | 第21-22页 |
| 1.6 Objective,purpose and content of this research | 第22-24页 |
| 2 Grafting between gouqi(Lycium chinense Mill.) and tomato | 第24-30页 |
| 2.1 Materials,methods,reagents and equipment | 第24页 |
| 2.1.1 Plant materials | 第24页 |
| 2.1.2 Non-plant materials | 第24页 |
| 2.2 Mother plant preparation for grafting | 第24-25页 |
| 2.2.1 Preparation of root stock mother plant/control gouqi plants | 第24-25页 |
| 2.2.2 Preparation of scion mother plant/control tomato plants | 第25页 |
| 2.3 Grafting method and procedure | 第25-27页 |
| 2.3.1 Grown up of grafted plants | 第25页 |
| 2.3.2 Target tissue selection | 第25-27页 |
| 2.4 Total RNA isolation | 第27页 |
| 2.4.1 Materials and equipment | 第27页 |
| 2.4.2 Chemicals and reagents | 第27页 |
| 2.5 Methods | 第27-29页 |
| 2.5.1 Harvesting target tissues | 第27页 |
| 2.5.2 Homogenization | 第27-28页 |
| 2.5.3 Phase separation | 第28页 |
| 2.5.4 RNA precipitation | 第28页 |
| 2.5.5 RNA wash | 第28页 |
| 2.5.6 Re-dissolving the RNA | 第28页 |
| 2.5.7 Quantification and quality control | 第28-29页 |
| 2.6 Total RNA isolation from shoot | 第29页 |
| 2.7 Total RNA isolation from fruit | 第29-30页 |
| 3 High-throughput RNA sequencing | 第30-37页 |
| 3.1 Materials and methods | 第30-32页 |
| 3.1.1 Sample tissues | 第30-31页 |
| 3.1.2 Equipment | 第31页 |
| 3.1.3 Quality check for RNA isolation for sequencing | 第31页 |
| 3.1.4 Small RNA library preparation | 第31-32页 |
| 3.2 Downstream bioinformatics | 第32-37页 |
| 3.2.1 Clustering and sequencing | 第32页 |
| 3.2.2 Data analysis | 第32-37页 |
| 3.2.2.1 Quality control | 第32页 |
| 3.2.2.2 Reads mapping to the reference sequence | 第32-33页 |
| 3.2.2.3 Evolutionary Known miRNA alignment | 第33页 |
| 3.2.2.4 Remove tags from these sources | 第33-34页 |
| 3.2.2.5 Novel miRNA prediction | 第34页 |
| 3.2.2.6 Summary of small RNA annotation | 第34页 |
| 3.2.2.7 miRNA editing analysis | 第34页 |
| 3.2.2.8 miRNA family analysis | 第34页 |
| 3.2.2.9 Target gene prediction | 第34-35页 |
| 3.2.2.10 Quantification of miRNA | 第35页 |
| 3.2.2.11 Differential expression of miRNA | 第35页 |
| 3.2.2.12 Functional assignments of potential target genes | 第35页 |
| 3.2.2.13 Validation of miRNA expression using qRT-PCR | 第35-36页 |
| 3.2.2.14 Validation of predicted targets using RLM 5'-RACE | 第36-37页 |
| 4 Small RNA profiling in gouqi | 第37-54页 |
| 4.1 Background | 第37页 |
| 4.1.1 Objectives | 第37页 |
| 4.2 Results | 第37-47页 |
| 4.2.1 Summary of small-RNA library dataset by deep sequencing in gouqi | 第37-39页 |
| 4.2.2 Identification of conserved and evolutionarily known miRNAs | 第39-40页 |
| 4.2.3 Prediction of potentially novel miRNAs and nucleotide bias | 第40-41页 |
| 4.2.4 Differentially expressed known and novel miRNAs between the shoot and fruit | 第41-45页 |
| 4.2.5 Prediction and annotation of potential target genes of differentially expressed miRNAs | 第45-47页 |
| 4.3 Expression validation of sequencing data with qRT-PCR and RLM 5'-RACE | 第47-49页 |
| 4.4 Discussion | 第49-53页 |
| 4.4.1 Overview of the deep-sequencing datasets | 第49-51页 |
| 4.4.2 Target gene annotation of fruit-related miRNAs in L. chinense | 第51-52页 |
| 4.4.3 Target gene annotation of shoot-specific miRNAs in L. chinense | 第52-53页 |
| 4.5 Conclusion | 第53-54页 |
| 5 Comparative profiling of small RNAs in control and grafted tomato | 第54-82页 |
| 5.1 Background | 第54页 |
| 5.1.1 Objectives | 第54页 |
| 5.2 Results | 第54-68页 |
| 5.2.1 Summary of small-RNA library dataset by deep sequencing in the grafted and control tomato | 第54-58页 |
| 5.2.2 Identification of evolutionary known miRNAs | 第58-60页 |
| 5.2.3 Prediction of potentially novel miRNAs and nucleotide bias | 第60-66页 |
| 5.2.4 Differentially expressed known and potentially novel miRNAs between grafted and control tomato | 第66-68页 |
| 5.3 Target genes annotation; GO and KEGG analysis of grafted tomato fruits | 第68-71页 |
| 5.4 Expression validation of sequencing data with qRT-PCR and RLM 5'-RACE | 第71-73页 |
| 5.5 Discussion | 第73-81页 |
| 5.5.1 miRNAome in shoot and fruit tissues of grafted and control tomato | 第74-76页 |
| 5.5.2 Target annotation of differentially expressed miRNAs | 第76-81页 |
| 5.6 Conclusion | 第81-82页 |
| 6 Identification and analysis of miRNA transported from gouqi to tomato | 第82-84页 |
| References | 第84-96页 |
| Appendices | 第96-116页 |
| Student Profile | 第116-118页 |
| Acknowledgements | 第118页 |
