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利用CRISPR/Cas9编辑水稻木质素单体合成途径基因

摘要第6-7页
ABSTRACT第7页
ABBREVIATION第8-10页
1 INTRODUCTION第10-30页
    1.1 Literature review第13-29页
        1.1.1 Rice as a Model Plant第13-14页
        1.1.2 Lignin and Lignocellulosic Biomass Conversion第14-15页
        1.1.3 The Lignin Monolignol Biosynthetic Pathway第15-17页
        1.1.4 The Recent Developments in Transgenic Technologies and CRISPR/Cas9 Gene-Editing System第17-29页
    1.2 Research Objectives第29-30页
2 MATERIAL AND METHOD第30-51页
    2.1 Selection of Genes第30页
    2.2 Designing sgRNA第30-32页
    2.3 Vector Construction第32-34页
        2.3.1 Primer Renaturation第32-33页
        2.3.2 CRISPR Vector Restriction Digestion第33页
        2.3.3 DNA and Vector Ligation第33-34页
    2.4 Transform Escherichia coli DH5α Competent Cells第34-37页
        2.4.1 Preparation of DH5α Competent Cells第34-36页
        2.4.2 Transforming DH5α Competent Cells第36页
        2.4.3 LB-Medium第36-37页
    2.5 Selection of Positive Colonies第37-39页
        2.5.1 PCR Protocol第37-39页
        2.5.2 Sanger Sequencing第39页
    2.6 Sub-Clone Vector into Agrobacterium第39-40页
        2.6.1 Freeze–Thaw Method第40页
    2.7 Rice Transformation第40-47页
        2.7.1 Induction of Calli第41页
        2.7.2 Preparation of Calli and Agrobacterium第41页
        2.7.3 Infection of Callus with Agrobacterium第41-47页
    2.8 Detection of Positive Plants第47-50页
        2.8.1 DNA Extraction第47-49页
        2.8.2 CTAB Extraction Buffer第49-50页
    2.9 Software and Websites Used第50-51页
3 RESULTS AND ANALYSIS第51-84页
    3.1 Collection of Protein Sequences and Alignment第51页
    3.2 Identities of Genes with their Homologous Proteins第51-54页
    3.3 Multiple Sequence Alignment and Phylogenetic Tree第54-59页
    3.4 3D Proteins Structures Prediction第59-62页
    3.5 Identification of Unique Sites in Proteins to Design sgRNA第62-63页
    3.6 Designed sgRNA第63页
    3.7 Vector Construction第63-67页
    3.8 Identification of Positive E. coli Colonies and Sequencing Results第67-69页
    3.9 Sub-Cloning of Plasmid into Agrobacterium tumefaciens第69-71页
    3.10 T0 Generation Mutants第71-73页
    3.11 Mutations Observed in T0 Generation第73-75页
    3.12 Phenotypes Observed in Mutant Plants第75-78页
    3.13 Mutants Protein Sequence Predictions and Alignment第78-84页
4 DISCUSSION第84-87页
    4.1 Some Remaining Issues第86-87页
REFERENCES第87-104页
Acknowledgements第104-105页

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