| Abstract | 第1-7页 |
| 摘要 | 第7-8页 |
| Acronyms | 第8-12页 |
| SECTION 1: Literature Review | 第12-26页 |
| ·Introduction | 第12-14页 |
| ·Identification of M. oryzae | 第14-15页 |
| ·Disease cycle | 第15-16页 |
| ·Deducing pathogenicity determinants in the life cycle of M. oryzae | 第16-18页 |
| ·M. oryzae pre-mRNA splicing pathways | 第18-20页 |
| ·Alternate RNA forms and alternative splicing in M. oryzae | 第20-21页 |
| ·BBP gene and its corresponding BPS | 第21-22页 |
| ·mRNA interference in M. oryzae | 第22-24页 |
| ·Objective of the study | 第24-25页 |
| ·Research areas | 第25-26页 |
| SECTION 2: Knockout of the BBP gene | 第26-49页 |
| ·Materials and Methods | 第26页 |
| ·Materials | 第26-32页 |
| ·Bacteria, fungal strain and plasmids | 第26页 |
| ·Major experimental kits and reagents | 第26页 |
| ·Major experimental equipments and machines | 第26页 |
| ·Major formulas for experimental solution and media | 第26-32页 |
| ·Methods | 第32-44页 |
| ·Fungal strains cμltivation and growth conditions | 第32页 |
| ·CTAB Method for Fungal DNA extraction | 第32-39页 |
| ·Vector plasmid and BBP gene DNA manipμlations | 第33-34页 |
| ·PCR condition for each of the two flanks | 第34页 |
| ·PCR product recovering and ligation into pMD18-T vector | 第34-35页 |
| ·Procedure for Transformation into the E. coli DH5α competent cell | 第35页 |
| ·Extraction of plasmid | 第35-36页 |
| ·Confirmation of successful ligation | 第36-37页 |
| ·PCR confirmation | 第36-37页 |
| ·Double restriction endonuclease digestion | 第37页 |
| ·Sequencing | 第37页 |
| ·Construction of knockout vector | 第37-39页 |
| ·Insertion of the left flank | 第38-39页 |
| ·Transformation into E. coli competent cell | 第39页 |
| ·Insertion of the right flank | 第39页 |
| ·Transformation into E. coli competent cell | 第39页 |
| ·Confirmation for the successful ligation | 第39页 |
| ·Transformation of pXEH-BBP into Agrobacterium strain EHA10528 | 第39-40页 |
| ·Procedures for EHA105 transformation | 第39-40页 |
| ·Confirmation of successfμl tansformation | 第40页 |
| ·Agrobacterium mediated transformation of the M. oryzae strain JJ88 | 第40-41页 |
| ·Procedure for ATMT | 第40-41页 |
| ·Protoplast transformation | 第41-42页 |
| ·Procedures for protoplast transformation | 第41-42页 |
| ·Extraction of transformants DNA using lysis solution | 第42页 |
| ·Procedures for DNA extraction using lysis solution | 第42页 |
| ·Screening for the mutants from transformants by PCR | 第42-44页 |
| ·Explanation of screening for the mutants | 第42-44页 |
| ·Results | 第44-47页 |
| ·Amplification of the BBP left and right flanks | 第44页 |
| ·Construction of pXEH-BBP knockout vector | 第44-45页 |
| ·Transformation into Agrobacterium strain EHA105 | 第45-46页 |
| ·Transformation into rice blast fungus M. oryzae strain JJ8835 | 第46页 |
| ·Knockout mutants | 第46-47页 |
| ·Discusion | 第47-49页 |
| SECTION 3 RNA Interference Of BBP Gene | 第49-61页 |
| ·Materials and methods | 第49-55页 |
| ·Materials | 第49页 |
| ·Methods | 第49-55页 |
| ·Construction of RNA interference species | 第49页 |
| ·PCR conditions | 第49-50页 |
| ·PCR product recovering and purification | 第50页 |
| ·Ligation into pMD18-T vector | 第50页 |
| ·Transformation into the DH5α competent cell | 第50-51页 |
| ·Extraction of plasmid | 第51页 |
| ·Confirmation of successful ligation | 第51-52页 |
| ·PCR confirmation | 第51页 |
| ·Double restriction endonuclease digestion for sense fragment | 第51页 |
| ·Double restriction endonuclease digestion for antisense fragment | 第51-52页 |
| ·Confirmation of cloned RNA interference fragments by sequencing | 第52页 |
| ·Construction of BBP RNAi vectors | 第52-53页 |
| ·pSilent-1 plasmid restriction digestion and ligation of the flanks | 第53-55页 |
| ·ligation of the sense RNAi fragment to get pSilent-1BBPRNAiS RNA silencing species | 第53页 |
| ·Ligation | 第53页 |
| ·Transformation into E. coli competent cell | 第53页 |
| ·Confirmation of successful ligation | 第53-54页 |
| ·Ligation of the antisense fragment in pSilent-1BBPRNAiS to get hairpin RNA interference species | 第54页 |
| ·Ligation | 第54页 |
| ·Transformation into E. coli competent cell | 第54页 |
| ·Confirmation for the successfμl ligation | 第54-55页 |
| ·Transformation into M. oryzae strain JJ8844 | 第55页 |
| ·Protoplast transformation | 第55页 |
| ·Screeing for transformants | 第55页 |
| ·Assays for growth and sporulation | 第55页 |
| ·Results | 第55-59页 |
| ·Amplification of the BBP sense and antisense fragments | 第55-56页 |
| ·RNA interference species | 第56-57页 |
| ·Transformants | 第57页 |
| ·Assays for growth and sporulation | 第57-59页 |
| ·Discussions | 第59-61页 |
| SECTION 4 | 第61-62页 |
| ·Conclusion | 第61-62页 |
| References | 第62-70页 |
| Acknowledgments | 第70页 |
