| 摘要 | 第6-8页 |
| Abstract | 第8-9页 |
| Abbreviation | 第18-20页 |
| CHAPTER I GENERAL INTRODUCTION | 第20-42页 |
| 1.1 Rice is an important agricultural crop | 第20-22页 |
| 1.1.1 Rice in China | 第20-22页 |
| 1.1.2 Rice in Bangladesh | 第22页 |
| 1.2 History of rice breeding and as a model system for monocotyledonous plants | 第22-24页 |
| 1.3 Recurrent selection and gene pyramiding | 第24-25页 |
| 1.4 Genetic engineering | 第25-28页 |
| 1.4.1 Microbial vectors | 第26页 |
| 1.4.2 Microprojectile or particle bombardment | 第26页 |
| 1.4.3 Electroporation | 第26页 |
| 1.4.4 Microinjection | 第26页 |
| 1.4.5 Transposons/Transposable elements | 第26-27页 |
| 1.4.6 Common process of GE | 第27-28页 |
| 1.5 Gene silencing | 第28-39页 |
| 1.5.1 RNA interference | 第29-30页 |
| 1.5.2 Discovery of RNAi | 第30-32页 |
| 1.5.3 Designing RNAi vector for efficient silencing | 第32页 |
| 1.5.4 RNAi in plant improvement | 第32-35页 |
| 1.5.5 Vector and gene for efficient plant transformation to silence with RNAi | 第35-39页 |
| (a) RNAi vector for silencing | 第35页 |
| (b) Target the gene for silencing | 第35-39页 |
| (b.1) Silencing the dwarf trait | 第36-37页 |
| (b.2) Male-sterile gene for silencing of male fertility | 第37-39页 |
| 1.6 Transgenic plants, human health and safety | 第39-41页 |
| 1.7 Objectives of this study | 第41-42页 |
| CHAPTER II CONSTRUCTION OF p TCK-DIID RNAi VECTOR TO SILENCEOs GA20ox2 AND DTD GENE | 第42-57页 |
| 2.1 Summary | 第42页 |
| 2.2 Introduction | 第42-44页 |
| 2.3 Materials and methods | 第44-50页 |
| 2.3.1 Plant and experimental materials | 第44-46页 |
| 2.3.2 Primers design | 第46-48页 |
| 2.3.3 Vector and gene construct | 第48-49页 |
| 2.3.3.1 Construction of the antisense strand | 第48-49页 |
| 2.3.3.2 Construction of the sense strand | 第49页 |
| 2.3.4 Construction of hairpin RNAi vector named as p TCK-DIID | 第49-50页 |
| 2.4 Results | 第50-54页 |
| 2.5 Discussion | 第54-57页 |
| CHAPTER III GENETIC TRANSFORMATION OF RICE BY USING p TCK-RIIR ANDp TCK-DIID RNAi VECTORS | 第57-67页 |
| 3.1 Summary | 第57页 |
| 3.2 Introduction | 第57-59页 |
| 3.3 Materials and methods | 第59-61页 |
| 3.3.1 Plant material, other experimental materials and growth conditions | 第59-60页 |
| 3.3.2 Procedures of Agrobacterium-mediated genetic transformation of rice calli | 第60-61页 |
| 3.3.2.1 Embryogenic callus induction | 第60页 |
| 3.3.2.2 Callus subculture | 第60页 |
| 3.3.2.3 Precultivaton and culture of agrobacterium for infection | 第60页 |
| 3.3.2.4 Agrobacterium infection and co-cultivation | 第60-61页 |
| 3.3.2.5 Selection of callus | 第61页 |
| 3.3.2.6 Callus regeneration | 第61页 |
| 3.3.2.7 Growing plantlets | 第61页 |
| 3.4 Results | 第61-64页 |
| 3.4.1 Embryogenic callus induction and culture | 第61-62页 |
| 3.4.2 Inoculation, cultivation and selection of callus | 第62-63页 |
| 3.4.3 Plant regeneration and developing | 第63-64页 |
| 3.5 Discussion | 第64-67页 |
| CHAPTER IV MOLECULAR ANALYSIS OF TRANSGENIC RICE PLANTS | 第67-83页 |
| 4.1 Summary | 第67页 |
| 4.2 Introduction | 第67-69页 |
| 4.3 Materials and methods | 第69-74页 |
| 4.3.1 Plant material | 第69页 |
| 4.3.2 Evaluation of transgenic plants by PCR analysis | 第69-71页 |
| 4.3.2.1 Genomic DNA extraction method | 第69-70页 |
| 4.3.2.2 Evaluation of transgenic plants from p TCK-RIIR and p TCK-DIID vectors by PCR analysis withhygromycin paired primers | 第70页 |
| 4.3.2.3 Evaluation of p TCK-RIIR transgenic plants by PCR analysis with gene specific primers | 第70页 |
| 4.3.2.4 Evaluation of p TCK-DIID transgenic plants by PCR analysis with gene specific primers | 第70-71页 |
| 4.3.3 Gene expression strategy | 第71-74页 |
| 4.3.3.1 RNA preparation and reverse transcription of putative transgenic plants | 第71-72页 |
| 4.3.3.2 Evaluation of expression of transgenic plants by RT-PCR analysis | 第72-74页 |
| 4.3.3.3 Evaluation of expression of transgenic plants by q RT-PCR Analysis | 第74页 |
| 4.4 Results | 第74-80页 |
| 4.4.1 Evaluation of transgenic plants by PCR analysis | 第74-77页 |
| 4.4.2 Gene expression strategy | 第77-80页 |
| 4.4.2.1 RT-PCR analysis | 第77-79页 |
| 4.4.2.2 Quantitative RT-PCR analysis | 第79-80页 |
| 4.5 Discussion | 第80-83页 |
| CHAPTER V PHENOTYPIC ANALYSIS OF TRANSGENIC RICE PLANTS | 第83-110页 |
| 5.1 Summary | 第83页 |
| 5.2 Introduction | 第83-84页 |
| 5.3 Materials and methods | 第84-87页 |
| 5.3.1 Research facilities | 第84-85页 |
| 5.3.2 Plant material, other experimental materials and growth conditions | 第85页 |
| 5.3.3 Phenotyping of the generated transgenic RNAi plants | 第85-86页 |
| 5.3.3.1 Evaluation of transgenic rice for plant height | 第86页 |
| 5.3.3.2 Evaluation of transgenic rice for male sterility | 第86页 |
| 5.3.4 Production of Seeds of DMS lines and genetic cross | 第86-87页 |
| 5.3.5 Data analysis | 第87页 |
| 5.4 Results | 第87-108页 |
| 5.4.1 Phenotypic analysis in different progeny of p TCK-RIIR RNAi knockdown plants | 第88-95页 |
| 5.4.1.1 Identification of dwarf rice mutant by the suppression of Os GA20ox2 expression | 第88-91页 |
| 5.4.1.2 Identification of sterile rice mutant | 第91-93页 |
| 5.4.1.3 RNAi vectors contributed to other yield traits of DMS rice | 第93-95页 |
| 5.4.1.4 Transgene inheritance and generational stability of transgene expression in the different progenyby using p TCK-RIIR vector | 第95页 |
| 5.4.2 Phenotypic analysis of RNAi knockdown plants at the different progeny of p TCK-DIID inducedDMS rice | 第95-108页 |
| 5.4.2.1 Identification of dwarf rice mutant by silencing of Os GA20ox2 gene | 第95-99页 |
| 5.4.2.2 Identification of sterile rice mutant | 第99-103页 |
| 5.4.2.3 RNAi vectors contributed to other yield traits of dwarf and male sterile rice | 第103-106页 |
| (a) Plant height | 第103页 |
| (b) Number of effective tillers per plant | 第103-104页 |
| (c) Panicle length | 第104-105页 |
| (d) Spikelet per panicle | 第105页 |
| (e) Days to heading | 第105页 |
| (f) Days to maturity | 第105-106页 |
| 5.4.2.4 Transgene inheritance and generational stability of transgene expression in the different progenyof p TCK-DIID-induced DMS rice | 第106-108页 |
| 5.5 Discussion | 第108-110页 |
| CHAPTER VI GENE TRANSFER BY USING DMS RICE | 第110-123页 |
| 6.1 Summary | 第110页 |
| 6.2 Introduction | 第110-112页 |
| 6.3 Materials and methods | 第112-114页 |
| Statistical analysis | 第113-114页 |
| 6.4 Results | 第114-121页 |
| 6.4.1 Phenotypic analysis of RNAi knockdown plants at the different progeny | 第114-120页 |
| 6.4.1.1 Identification of dwarf rice mutants | 第114-115页 |
| 6.4.1.2 Identification of sterile rice mutants | 第115-118页 |
| 6.4.1.3 RNAi vectors contributed to other yield traits of DMS rice | 第118-120页 |
| 6.4.2 Transgene inheritance and generational stability of transgene expression in the different progenyby using p TCK-DIID vector | 第120-121页 |
| 6.5 Discussion | 第121-123页 |
| GENERAL CONCLUSION | 第123-126页 |
| Recommendations | 第125-126页 |
| REFERENCES | 第126-141页 |
| ACKNOWLEDGEMENTS | 第141-143页 |
| RESUME | 第143-150页 |
| APPENDIX List of gene sequences relate with the RNAi vector | 第150-157页 |
