| 摘要 | 第1-4页 |
| Abstract | 第4-8页 |
| 1 Introduction | 第8-23页 |
| ·Hypovirus and Chestnut Blight Fungus | 第8-14页 |
| ·Biology of Chestnut Blight Fungus | 第8-9页 |
| ·Molecular Genetics of Hypovirus | 第9-12页 |
| ·Interaction between Hypovirus and the Fungal Host, Cryphonectria parasitica | 第12-14页 |
| ·Shuttle Plasmid | 第14-21页 |
| ·Prokaryotic Shuttle Vector | 第14-15页 |
| ·Autonomously Replicating Plasmids in Fungi | 第15-21页 |
| ·Plasmids in Yeast | 第15-17页 |
| ·Autonomously Replicating Plasmids in Filamentous Fungi | 第17-21页 |
| ·Genetic Manipulation System of C.parasitica | 第21-23页 |
| ·DNA-mediated Transformation | 第21页 |
| ·Integrative Vectors for C.parasitica | 第21页 |
| ·Viral Transfection for C.parasitica | 第21-22页 |
| ·Plasmids in C.parasitica | 第22-23页 |
| 2 Materials and Methods | 第23-28页 |
| ·Materials | 第23-24页 |
| ·Fungal Strains and Media | 第23页 |
| ·Bacterial Strains and Plasmids | 第23页 |
| ·Enzymes, Reagents and Kits | 第23-24页 |
| ·Methods | 第24-28页 |
| ·Bacterial Culture | 第24页 |
| ·Plasmid Preparation | 第24页 |
| ·Anastomosis Test | 第24-25页 |
| ·Transformation of C.parasitica EP713 Spheroplasts | 第25页 |
| ·Chromosome DNA Preparation | 第25页 |
| ·Total RNA Isolation | 第25页 |
| ·Molecular Manipulations | 第25-26页 |
| ·Restriction, Ligation, and Transformation | 第25页 |
| ·PCR and RT-PCR | 第25页 |
| ·Southern Hybridyzation Analysis | 第25-26页 |
| ·Laccase Activity Assay | 第26-27页 |
| ·Strategy for Plasmid Optimization | 第27-28页 |
| 3 Results | 第28-55页 |
| ·pGXH1130 Replicates in the Cytoplasm of C.parasitica | 第28-29页 |
| ·Optimization of pGXH1130 | 第29-38页 |
| ·Generation of pGXH△XhoI | 第29页 |
| ·Generation of pGXH△NheI | 第29-30页 |
| ·Generation of pGXH△(N+H) | 第30-31页 |
| ·Generation of pGXH△(N+X) | 第31-32页 |
| ·Generation of pGXH△(N+H+X) | 第32-33页 |
| ·Generation of pGXH4△ | 第33-35页 |
| ·Generation of pGXH4△-1 | 第35-36页 |
| ·Generation of pFC9 | 第36-37页 |
| ·RFLP Analysis of pGXH Vectors | 第37-38页 |
| ·Morphology of C.parasitica EP713 Transformed by pGXH Vectors | 第38-40页 |
| ·The Modes of Existence of pGXH Vectors in C.parasitica | 第40-43页 |
| ·pFC9 Functioned as An Expression Vector | 第43-52页 |
| ·Development of the Expression Construct pFC9-Lac1 | 第43-48页 |
| ·Cloning of LAC-1 Full Length cDNA | 第43-44页 |
| ·Generation of An Expression Construct pFC9-Lac1 | 第44-46页 |
| ·Expression of LAC-1 cDNA in C.parasitica EP713 | 第46-47页 |
| ·PCR Identification of the Transformants, EP713/pFC9-Lac1 | 第47-48页 |
| ·Development of the Expression Construct pFC9-gLac1 | 第48-52页 |
| ·Cloning of Genomic LAC-1 Gene | 第48-49页 |
| ·Generation of An Expression Construct, pFC9-gLac1 | 第49-50页 |
| ·Expression of Genomie LAC-1 in C.parasitica EP713 | 第50-51页 |
| ·PCR Detection of the Transformants, EP713/pFC9-gLac1 | 第51-52页 |
| ·Manner of Existence of pFC9 in the Cells of C.parasitica | 第52-54页 |
| ·Mobility of pFC9 in Hypha | 第54-55页 |
| 4 Discussions | 第55-58页 |
| ·Possible Autonomously Replicating Sequence (ARS) in pFC9 | 第55-56页 |
| ·Copy Numbers of pFC9 in C.parasitica | 第56页 |
| ·Potential of pFC9 | 第56-58页 |
| Acknowledgements | 第58-59页 |
| References | 第59-65页 |
| ADJUNCTS | 第65-70页 |
