| 摘要 | 第4-7页 |
| abstract | 第7-9页 |
| 1. Introduction | 第19-31页 |
| 1.1 Effecting of drought on plants | 第19-20页 |
| 1.2 Genetic modification improved drought tolerance in sugarcane | 第20-23页 |
| 1.2.1 Sugarcane conventional breeding situation | 第20-21页 |
| 1.2.2 Developments and application of transgenic technology on sugarcane | 第21-23页 |
| 1.3 Overview of SnRKs gene in plants | 第23-27页 |
| 1.3.1 Structural Analysis of SnRKs family | 第23-24页 |
| 1.3.2 Regulation and activity of SnRK2 gene in plants | 第24-26页 |
| 1.3.3 Functional of SnRK2 in plant | 第26-27页 |
| 1.4 Overview of ACLs gene in plants | 第27-29页 |
| 1.4.1 Mechanism of ACLs | 第27-28页 |
| 1.4.2 Functional of ACLs in plant | 第28-29页 |
| 1.5 Objective | 第29-31页 |
| 2. Cloning, prokaryotic expression, purification and function identify of SoACLA-1 andSoSnRK2.1 from sugarcane and preparation of antiserum | 第31-49页 |
| 2.1 Materials and methods | 第32-37页 |
| 2.1.1 Bacterial strain, media, and vector | 第32页 |
| 2.1.2 Main reagent and equipment | 第32页 |
| 2.1.3 Cloning the full-length SoACLA-1 and SoSnRK2.1 gene | 第32-33页 |
| 2.1.4 Construction of expression vectors for SoACLA-1 and SoSnRK2.1 genes | 第33-34页 |
| 2.1.5 Expression of the recombinant protein from E. coli | 第34-35页 |
| 2.1.6 Purification of the recombinant protein from E. coli | 第35-36页 |
| 2.1.7 Protein Concentration and monoclonal antibody preparation | 第36页 |
| 2.1.8 Recombinant under PEG treatment | 第36-37页 |
| 2.2 Results | 第37-46页 |
| 2.2.1 Isolation and identification of SoACLA-1 and SoSnRK2.1 genes | 第37-38页 |
| 2.2.2 Molecular characterization of SoACLA-1 and SoSnRK2.1 | 第38-41页 |
| 2.2.3 Construction of the pET-SoACLA-1 and pET-SoSnRK2.1 expression Vectors | 第41-42页 |
| 2.2.4 Expression and purification of pET-SoACLA-1 and pET-SoSnRK2.1 | 第42-45页 |
| 2.2.5 Concentration protein and preparation for monoclonal antibodies | 第45页 |
| 2.2.6 Epression of SoACLA1 and SoSnRK2.1 genes enhances PEG stress | 第45-46页 |
| 2.3 Discussion | 第46-49页 |
| 3. Overexpression of SoSnRK2.1 improved drought tolerance in tobacco | 第49-73页 |
| 3.1 Materials and method | 第49-56页 |
| 3.1.1 Plant material and bacteria stain | 第49页 |
| 3.1.2 Experimental equipments and reagents | 第49-50页 |
| 3.1.3 Transformation tissue cultures | 第50页 |
| 3.1.4 Transgenic vector construction | 第50-51页 |
| 3.1.5 Plant transformation and transgenic tobacco generation | 第51-52页 |
| 3.1.6 PCR analysis | 第52页 |
| 3.1.7 RT-PCR analysis expression of SoSnRK2.1 in transgenic tobacco | 第52-53页 |
| 3.1.8 Southern blot analysis | 第53页 |
| 3.1.9 Drought tolerance experiment of WT and transgenic tobacco plants | 第53-54页 |
| 3.1.10 Measurement and analysis of RWC, IL, MDA accumulation in WT and transgenic plants36 | 第54页 |
| 3.1.11. Measurement of SOD, POD and CAT activities, H_2O_2 and Chlorophyll content | 第54-55页 |
| 3.1.12 Sub-cellular Localization of SoSnRK2.1 | 第55页 |
| 3.1.13 Transgene inheritance experiment | 第55-56页 |
| 3.1.14 Statistical analysis | 第56页 |
| 3.2 Results | 第56-71页 |
| 3.2.1 Transgene construction | 第56-57页 |
| 3.2.2 Transgenic tobacco plants generation | 第57-59页 |
| 3.2.3 Detection of transgenic tobacco plants | 第59-61页 |
| 3.2.4 Over-expression of SoSnRK2.1 improves drought tolerance in transgenic plants | 第61-63页 |
| 3.2.5 Transgenic plants via over-expression of SoSnRK2.1 increased RWC and declined MDAand IL content under drought stress condition | 第63-65页 |
| 3.2.6 Over-expression of SoSnRK2.1 enhances chlorophyll content and reduces H_2O_2concentration under drought stress | 第65-66页 |
| 3.2.7 Over-expression of SoSnRK2.1 in transgenic tobacco plants increases activities of threeantioxidants enzymes under drought stress | 第66-68页 |
| 3.2.8 Transgenic plants inheritance experiment | 第68-70页 |
| 3.2.9 Over-expression of SoSnRK2.1 in T2 transgenic plants improves drought tolerance | 第70-71页 |
| 3.3 Discussion | 第71-73页 |
| 4. SoSnRK2.1 gene transferred in Sugarcane improving drought tolerance | 第73-98页 |
| 4.1 Materials and methods | 第73-79页 |
| 4.1.1 Plant material and bacteria stain | 第73-74页 |
| 4.1.2 Plasmid | 第74页 |
| 4.1.3 Experimental equipments and reagents | 第74页 |
| 4.1.4 Transformation tissue cultures conditions | 第74-75页 |
| 4.1.5 Expressed vector construction | 第75-77页 |
| 4.1.6 Tissue culture of sugarcane | 第77页 |
| 4.1.7 Kill curve experiment | 第77页 |
| 4.1.8 Agro bacterial infection, Co-cultivation and Sugarcane transformation | 第77-78页 |
| 4.1.9 Drought tolerance of the WT and sugarcane transgenic plants | 第78-79页 |
| 4.1.10 Statistical analysis | 第79页 |
| 4.2 Results | 第79-93页 |
| 4.2.1 Transgene construction of pRI-SoSnRK2.1 and its introduction into A. tumefaciens | 第79-80页 |
| 4.2.2 The kill curve of G418 of callus | 第80-82页 |
| 4.2.3 The kill curve of G418 of shoots | 第82-83页 |
| 4.2.4 The kill curve of G418 of roots | 第83-84页 |
| 4.2.5 Injection and co-culture and generation of sugarcane transformation by Agrobacterium | 第84-86页 |
| 4.2.6 Selection of putative transgenic sugarcane | 第86-89页 |
| 4.2.7 Molecular analysis of putative sugarcane transgenic | 第89-91页 |
| 4.2.8 Drought assay | 第91-93页 |
| 4.3 Discussion | 第93-98页 |
| 5. Overexpression of ACLA-1 gene from sugarcane and its enhanced drought tolerance intobacco transgenic tobacco | 第98-118页 |
| 5.1 Materials and methods | 第98-101页 |
| 5.1.1 Plant material and bacteria strain | 第98页 |
| 5.1.2 Experimental equipments and reagents | 第98-99页 |
| 5.1.3 Transformation tissue cultures | 第99页 |
| 5.1.4 Transgenic vector construction | 第99-100页 |
| 5.1.5 Regeneration of transgenic tobacco | 第100页 |
| 5.1.6 PCR analysis | 第100页 |
| 5.1.7 RT-PCR analysis expression of SoACLA-1 in transgenic tobacco | 第100-101页 |
| 5.1.8 Drought tolerance experiment of WT and transgenic tobacco plants | 第101页 |
| 5.1.9 Statistical Analysis | 第101页 |
| 5.2 Results | 第101-114页 |
| 5.2.1 Transgenic vector construction and tobacco transformation | 第101-105页 |
| 5.2.2 Transgene inheritance assay of SoACLA-1 transgenic tobacco | 第105-107页 |
| 5.2.3 Drought stress assay for overexpresion of SoACLA-1 gene improved drought tolerance | 第107-108页 |
| 5.2.4 Overexpresion of SoACLA-1 gene enhances the RWC and decreases MDA and IL contentunder drought stress | 第108-111页 |
| 5.2.5 Overexpression of SoACLA-1 improved the activities of SOD,POD and CAT underdrought stress | 第111-112页 |
| 5.2.6 Overexpression of SoACLA-1 affect the contents of chlorophyll and H_2O_2 in sensetransgenic plants under drought stress | 第112-114页 |
| 5.3 Discussion | 第114-118页 |
| 6. ACLA gene transfer mediated by Agrobacterium tumefaciens enhances drought tolerance insugarcane | 第118-134页 |
| 6.1 Materials and methods | 第118-122页 |
| 6.1.1 Plasmid and bacteria strain | 第118页 |
| 6.1.2 Plants material | 第118-119页 |
| 6.1.3 Experimental equipments and reagents | 第119页 |
| 6.1.4 Culture condition | 第119页 |
| 6.1.5 Kill curve experiment | 第119-120页 |
| 6.1.6 Transgenic vector construction | 第120-121页 |
| 6.1.7 Preparation for A. tumefaciens suspension | 第121页 |
| 6.1.8 Sugarcane transformation and selection assay | 第121-122页 |
| 6.1.9 Analysis of expression of SoACLA-1 in transgenic sugarcane by PCR and RT-PCR | 第122页 |
| 6.1.10 Drought tolerance analysis of WT and sugarcane transgenic plants | 第122页 |
| 6.1.11 Statistical analysis | 第122页 |
| 6.2 Results | 第122-132页 |
| 6.2.1 The construction of pUBTC-SoACLA-1-GFP and its introduction into A. tumefaciensstrain | 第122-124页 |
| 6.2.2 The kill curve of PPT for shoots and roots | 第124-126页 |
| 6.2.3 Sugarcane transformation system mediated by A. tumefaciens strain | 第126-128页 |
| 6.2.4 Sugarcane transformation PCR analysis | 第128-131页 |
| 6.2.5 SoACLA-1 transgenic plants improve tolerance drought under drought stress condition | 第131-132页 |
| 6.3 Discussion | 第132-134页 |
| 7. Conclusions and prospects | 第134-138页 |
| 7.1 Conclusion | 第134-136页 |
| 7.2 Innovation points | 第136页 |
| 7.3 Suggestions and future prospects | 第136-138页 |
| References | 第138-162页 |
| Acknowledgements | 第162-163页 |
| Publication | 第163-164页 |
| Supplement Table | 第164-165页 |
