| 摘要 | 第5-7页 |
| Abstract | 第7-8页 |
| CHAPTER Ⅰ LITERATURE REVIEW | 第15-27页 |
| 1.1 Discovery and impact of bacterial blight disease (Xanthomonas oryzae pv. oryzae) | 第15-16页 |
| 1.2. Nitrogen metabolism and plant pathology | 第16-19页 |
| 1.2.1 The devastating biological environment caused by excess of nitrogen fertilizer | 第16-17页 |
| 1.2.2 Root nitrate transporters | 第17-18页 |
| 1.2.3 Nitrogen-limitation-induce early senescence | 第18-19页 |
| 1.3 Overlapping genes response to Xanthomonas oryzae pv. oryzae infection and N-limited condition | 第19页 |
| 1.4 Characterization of spotted leaf 11 (spl11 mutant) | 第19-21页 |
| 1.4.1 Arm repeat proteins | 第19-20页 |
| 1.4.2 Characterization of OsSPL11 in plant immunity and flowering | 第20-21页 |
| 1.5 NB-ARC domain containing resistance proteins in rice | 第21页 |
| 1.6 Detection of protein-protein interactions | 第21-26页 |
| 1.6.1 Yeast two-hybrid assay | 第22-24页 |
| 1.6.1.1 Matchmaker GAL4 Two-hybrid system | 第22-23页 |
| 1.6.1.2 Three different binding sites | 第23-24页 |
| 1.6.2 Confirming yeast two-hybrid protein interactions using in vitro glutathione-S-transferase pull-downs | 第24-25页 |
| 1.6.3 Firefly luciferase complementation imaging assay for protein-protein interactions in plants | 第25-26页 |
| 1.7 Objectives | 第26-27页 |
| CHAPTER Ⅱ:SPOTTED LEAF 11 (OSSPL11) CONTRIBUTES TO THE ADAPTATION TONITROGEN LIMITING IN RICE | 第27-41页 |
| 2.1 Introduction | 第27-28页 |
| 2.1.1 Regulation of root architecture under low nitrogen availability | 第27-28页 |
| 2.2 Materials and Methods | 第28-30页 |
| 2.2.1 Hydroponic condition for seedling growth | 第28-29页 |
| 2.2.2 qRT-PCR analysis | 第29页 |
| 2.2.3 Nitrate concentration | 第29页 |
| 2.2.4 Statistical analysis | 第29-30页 |
| 2.3 Results | 第30-39页 |
| 2.3.1 Expression pattern of OsSPL11 in different organs of the wild type and over expressing plants | 第30页 |
| 2.3.2 Reduce biomass accumulation in spil11 mutant under N-limited condition | 第30-33页 |
| 2.3.3 OsSPL11 regulate root growth and development under different nitrogen condition | 第33-36页 |
| 2.3.4 OsSPL11 effect the NRT gene expression pattern under N-limited condition | 第36-39页 |
| 2.4 Discussion | 第39-41页 |
| CHAPTER Ⅲ SPOTTED LEAF 11 (OSSPL11) WITHSTANDNITROGEN-LIMITATION-INDUCED LEAF SENESCENCE | 第41-53页 |
| 3.1 Introduction | 第41-42页 |
| 3.1.1 Nitrogen-limitation-induce leaf senescence | 第41-42页 |
| 3.1.2 Defense response and leaf senescence | 第42页 |
| 3.2 Materials and Methods | 第42-43页 |
| 3.2.1 Hydroponic condition for seedling growth | 第42-43页 |
| 3.2.2 qRT-PCR analysis | 第43页 |
| 3.2.3 Statistical analysis | 第43页 |
| 3.3 Results | 第43-50页 |
| 3.3.1 The precocious leaf senescence in spill mutants under N-limited condition | 第43-45页 |
| 3.3.2 Expression pattern of senescence-associated genes | 第45-46页 |
| 3.3.3 Nitrogen limitation stress derepresses OsSPL11-blocked defense-associated and cell death-associated gene expression under HN condition | 第46-50页 |
| 3.4 Discussion | 第50-53页 |
| 3.4.1 OsSPL11 regulates leaf senescence under both N-optimum and N-limited condition | 第50-51页 |
| 3.4.2 OsSPL11 plays a role in regulation of defense and cell death-associated gene expression under N-limited condition | 第51-53页 |
| CHAPTER Ⅳ CHARACTERIZATION OF OSRP-1 | 第53-74页 |
| 4.1 Introduction | 第53-54页 |
| 4.2 Materials and Methods | 第54-62页 |
| 4.2.1 Bioinformatics analysis of OsRP-1 | 第54页 |
| 4.2.2 Plant materials and stress treatment | 第54页 |
| 4.2.3 qRT-PCR analysis of OsRP-1 expression patterns | 第54页 |
| 4.2.4 Construction of rice cDNA library fusion vector | 第54页 |
| 4.2.5 Construction of pGBKT7:OsRP-1 bait fusion vector | 第54-59页 |
| 4.2.5.1 Total RNA extraction | 第55页 |
| 4.2.5.2 Single-strand cDNA synthesis | 第55-56页 |
| 4.2.5.3 PCR amplification of OsRP-1 gene | 第56页 |
| 4.2.5.4 Gel purification of PCR products | 第56-57页 |
| 4.2.5.5 Ligation procedure for vector construction | 第57页 |
| 4.2.5.6 Preparation of E.coli DH5α competent cells | 第57页 |
| 4.2.5.7 Transformation of pGEMT:OsRP1 into E.coli | 第57-58页 |
| 4.2.5.8 Extraction of plasmids for positive colony | 第58-59页 |
| 4.2.5.9 Verification of positive cloning vector | 第59页 |
| 4.2.6 Ligation of OsRP-1 to pGBKT7 bait fusion vector | 第59-60页 |
| 4.2.7 Transformation of pGBKT7:OsRP-1 into yeast Y2H Gold cells | 第60页 |
| 4.2.8 Two-Hybrid library screening using yeast mating | 第60-61页 |
| 4.2.9 Yeast colony PCR analysis to eliminate duplicate clones | 第61页 |
| 4.2.10 Annotation and gene ontology (GO) classification | 第61页 |
| 4.2.11 Confirmation of positive interactors | 第61-62页 |
| 4.2.11.1 Retransformation | 第61-62页 |
| 4.3 Results | 第62-73页 |
| 4.3.1 Phylogenetic analysis of OsRP-1 | 第62-63页 |
| 4.3.2 Analysis of the OsRP-1 sequence | 第63-64页 |
| 4.3.3 Expression of OsRP-1 is down-regulated in response to Xoo | 第64-65页 |
| 4.3.4 Screening potential interactors of OsRP-1 in rice | 第65-73页 |
| 4.3.4.1 Construction of rice cDNA library | 第65-66页 |
| 4.3.4.2 Uncut library and yeast library construction | 第66-67页 |
| 4.3.4.3 Characterization of Y2H cDNA library | 第67页 |
| 4.3.4.4 Construction of OsRP-1 bait fusion vector | 第67-68页 |
| 4.3.4.5 Verification of pGBKT7:OsRP-1 expression in Y2H Gold cells | 第68-70页 |
| 4.3.4.6 Investigation of interactors by library screen | 第70页 |
| 4.3.4.7 Annotation of bioinformatics and gene ontology classification | 第70-72页 |
| 4.3.4.8 Confirmation of positive interactors in yeast | 第72-73页 |
| 4.4 Discussion | 第73-74页 |
| CHAPTER Ⅴ CONFIRMATION OF THE INTERACTION BETWEEN OSRP-1 AND OSAHP2 | 第74-85页 |
| 5.1 Introduction | 第74-75页 |
| 5.1.1 Luciferase complementary imaging assay on Nicotiana benthamiana | 第74-75页 |
| 5.2 Materials and Methods | 第75-79页 |
| 5.2.1 GST pull-down | 第75-76页 |
| 5.2.2 Western blotting analysis | 第76-77页 |
| 5.2.2.1 Running, transfer, and blocking buffer | 第76-77页 |
| 5.2.3 Loading and running gel by SDS-PAGE | 第77-79页 |
| 5.2.2.0 Firefly Luciferase complementation imaging assay (LCI) | 第77-78页 |
| 5.2.3.1 Construction of split luciferase vectors | 第78页 |
| 5.2.3.2 Preparation of Agrobacterium competent cell | 第78页 |
| 5.2.3.3 Transformation of OsRP-1-NLuc and CLuc-OsAHP2 plasmid to Agrobacterium competent cells | 第78-79页 |
| 5.2.3.4 Agrobacterium-mediated transient expression | 第79页 |
| 5.3 Results | 第79-84页 |
| 5.3.1 Construction of bait protein and prey fusion vectors | 第79-84页 |
| 5.3.1.1 Cloning of pGEX-6p-1 bait fusion vector | 第79-80页 |
| 5.3.1.2 Cloning of pCold-Summo prey fusion vector | 第80页 |
| 5.3.1.3 Protein purification of GST fusion protein and His fusion protein | 第80-81页 |
| 5.3.1.4 GST pull-down confirmation of protein-protein interaction | 第81-83页 |
| 5.3.1.5 Verification of OsRP-1-NLuc and CLuc-OsAHP2 constructs | 第83页 |
| 5.3.1.5 Interaction between OsPR-1 and OsAHP2 in N. benthamiana | 第83-84页 |
| 5.4 Discussion | 第84-85页 |
| CHAPTER Ⅵ ANALYSIS ON FUNCTIONAL DOMAIN OF OSRP-1 | 第85-88页 |
| 6.1 Introduction | 第85页 |
| 6.2 Materials and Methods | 第85-86页 |
| 6.2.1 Plasmid constructs | 第85页 |
| 6.2.2 GST pull-down assay | 第85-86页 |
| 6.3 Results | 第86-87页 |
| 6.3.1 The CC and NBS Domains can mediate protein interaction | 第86-87页 |
| 6.4 Discussion | 第87-88页 |
| CHAPTER Ⅶ OSAHPS NEGATIVELY REGULATE PLANT DEFENSE AGAINSTXANTHOMONAS ORYZAE PV. ORYZAE | 第88-99页 |
| 7.1 Introduction | 第88-90页 |
| 7.1.1 Cytokinin signaling pathway | 第88-89页 |
| 7.1.2 Salicylic acid act synergistically with cytokinin to activate defense related gene expression in rice | 第89页 |
| 7.1.3 Protein-protein interaction of OsAHPs and other proteins | 第89-90页 |
| 7.2 Materials and Methods | 第90-92页 |
| 7.2.1 Hydroponic condition for seedling growth | 第90页 |
| 7.2.2. Genotyping of OsAHP2-RNAi lines from wild type | 第90-91页 |
| 7.2.3 Preparation of Xanthomonas oryzae pv. oryzae | 第91页 |
| 7.2.4 qRT-PCR analysis | 第91-92页 |
| 7.2.5 Statistical analysis | 第92页 |
| 7.3 Results | 第92-98页 |
| 7.3.1 OsAHPs-RNAi lines showed reduced plant height and internodes length | 第92-94页 |
| 7.3.2 OsAHPs-RNAi lines reduce the susceptibility to Xanthomonas oryzae pv. oryzae | 第94-95页 |
| 7.3.3 Defense-related gene expression in OsAHPs-RNAi-Xanthomonas oryzae pv. oryzae interaction | 第95-98页 |
| 7.4 Discussion | 第98-99页 |
| SUMMARY | 第99-100页 |
| REFERENCES | 第100-113页 |
| ACKNOWLEDGEMENT | 第113-115页 |
| TABLE 1 BACTERIAL STRAINS AND PLASMIDS USED IN THIS STUDY | 第115-116页 |
| TABLE 2 PRIMER USED IN THIS STUDY | 第116-117页 |
| TABLE 3 PRIMER USED IN THIS STUDY | 第117-118页 |
| TABLE 4 PUTATIVE PROTEINS FROM Y2H SCREENING RESULTS | 第118-124页 |
| AUTHOR BIOGRAPHY | 第124-125页 |
