| Abstract | 第1-8页 |
| 摘要 | 第8-11页 |
| Introduction | 第11-14页 |
| Overview of works | 第14-20页 |
| Part one:Expressional analysis of AvHMGR,AvDXS and AvDXR genes | 第20-28页 |
| ·Introduction | 第20-21页 |
| ·Material and methods | 第21-23页 |
| ·Plant materials | 第21页 |
| ·Sample preparation | 第21-22页 |
| ·RNA extraction | 第22页 |
| ·Semi-quantitative RT-PCR | 第22-23页 |
| ·Result and analysis | 第23-25页 |
| ·Total RNA extraction and analysis | 第23-24页 |
| ·Expressional analysis of AvHMGR,AvDXS and AvDXR in different samples of Ammomumvillosum | 第24-25页 |
| ·Discussion | 第25-26页 |
| ·Summary | 第26-28页 |
| Part two:Functional analysis of AvHMGR,AvDXS and AvDXR in E. coli cells | 第28-43页 |
| ·Introduction | 第28-29页 |
| ·Materials and methods | 第29-34页 |
| ·Bacteria and plasmids | 第29页 |
| ·Preparation of competent E.coli TOP10 cells | 第29-30页 |
| ·Extraction of pTrc-AtIPI plasmids from E.coli TOP10 cells | 第30页 |
| ·Amplification of AvHMGR,AvDXS and AvDXR fragments | 第30-31页 |
| ·Construction of recombinant plasmids pTrc-AvDXR | 第31-32页 |
| ·Enzyme digestion of AvDXR fragment and pTrc-AtIPI plasmids | 第31页 |
| ·Ligation and transformation | 第31-32页 |
| ·Screening of transformed E. coli TOP 10 cells | 第32页 |
| ·Construction of recombinant plasmids pTrc-AvHMGR and pTrc-AvDXS | 第32-33页 |
| ·Enzyme digestion of AvHMGR,AvDXS fragments and pTrc-AvDXR plasmids | 第32页 |
| ·Ligation and transformation | 第32-33页 |
| ·Screening of transformed E. coli TP10 cells | 第33页 |
| ·Construction of blank plasmid pTrc-SO | 第33页 |
| ·Construction of genetically engineered E. coli TOP10 cells | 第33-34页 |
| ·Generation of metabolic product by engineered E. coli TOP10 cells | 第34页 |
| ·Result and analysis | 第34-40页 |
| ·Generation of recombinant plasmids pTrc-AvHMGR,pTrc-AvDXS and pTrc-AvDXR | 第34-37页 |
| ·Preparation of negative control plasmid pTrc-SO | 第37-38页 |
| ·Funtional analysis of AvHMGR,AvDXS and AvDXS in E. coli | 第38-40页 |
| ·Discussion | 第40-42页 |
| ·Summary | 第42-43页 |
| Part three:Construction of transgenic tobacco plants using HMGR and DXR genes from A.villosum Lour | 第43-60页 |
| ·Introduction | 第43-44页 |
| ·Materials and methods | 第44-51页 |
| ·Plant materials | 第44页 |
| ·Bacteria and plasmids | 第44页 |
| ·Construction of binary vectors pBI-AvHMGR and pBI-AvDXR | 第44-46页 |
| ·Preparation of competent E. coil DH5α cells | 第44页 |
| ·Amplification of AvHMGR and AvDXR fragments | 第44-45页 |
| ·Extraction of binary vectors pBI-121 | 第45页 |
| ·Enzyme digestion of AvHMGR,AvDXR fragments and pBI-121 plasmids | 第45页 |
| ·Ligation and transformation | 第45-46页 |
| ·Screening of transformed E. coil cells | 第46页 |
| ·Construction of plant expression vectors | 第46-48页 |
| ·Preparation of competent A. tumifaciens cells | 第46-47页 |
| ·Transformation of recombinant plasmids into competent A.tumifaciens cells | 第47-48页 |
| ·Co-transformation of both recombinant plasmids into competent A.tumifaciens cells | 第48页 |
| ·Screening of transformed A.tumefaciens cells | 第48页 |
| ·Plant regeneration and transformation | 第48-50页 |
| ·Preparation of tobacco plants | 第48-49页 |
| ·Pre-culture of tobacco leaves | 第49页 |
| ·Preparation of transformed A.tumifaciens cells. | 第49页 |
| ·Infection of tobacco leaf with transformed A. tumefaciens cells | 第49-50页 |
| ·Screening of transgenic tobacco plants | 第50页 |
| ·Semi-quantitative RT-PCR analysis of transgenic plant | 第50-51页 |
| ·Result and analysis | 第51-57页 |
| ·Construction of recombinant plasmids | 第51-53页 |
| ·Construction of plant expression vectors | 第53-54页 |
| ·Generation of transgenic tobacco plant | 第54-55页 |
| ·Screening and RT-PCR analysis of AvHMGR and AvDXR in tobacco plants | 第55-57页 |
| ·Discussion | 第57-58页 |
| ·Summary | 第58-60页 |
| Summary of whole thesis | 第60-61页 |
| Acknowledgements | 第61-62页 |
| References | 第62-71页 |
