Clustered regularly interspaced short palindromic repeat-associated endonuclease Cas9（CRISPR/Cas9）is a novel genome editing technology that has recently been used extensively.The CRISPR/Cas9 system is derived from the adaptive immune system of Streptococcus thermophilus which is evolved against exogenous viruses and plasmids.It has been applied to various bio-related fields because of simplicity,high efficiency,and specificity.Although CRISPR/Cas9 system has been used in a variety of plants,the ... 

【文章來源】：海南大學(xué)海南省 211工程院校

【文章頁數(shù)】：59 頁

【學(xué)位級別】：碩士

【文章目錄】：
Abstract
1 Introduction
    1.1 Peanut
    1.2 High oleic acid
    1.3 FAD2 gene
    1.4 Conventional breeding method
    1.5 Transgenic approach
    1.6 Artificial nuclease
        1.6.1 ZFNs
        1.6.2 TALENs
    1.7 CRISPR/Cas system
        1.7.1 CRISPR structure
        1.7.2 Cas gene
        1.7.3 CRISPR/Cas classification
        1.7.4 The operational principle of CRISPR/Cas system
    1.8 CRISPR/Cas9-mediated genome modification
    1.9 Applications
        1.9.1 Genome editing
        1.9.2 Transcription regulation
    1.10 Challenges
    1.11 Research significance
    1.12 Technical structure
2 Materials and Methods
    2.1 Plant materials
    2.2 Agrobacterium strain and vector
    2.3 The construction of CRISPR/Cas9 vector
    2.4 Protoplast transformation
        2.4.1 Isolation of protoplasts from peanut leaves
        2.4.2 PEG-mediated transformation of protoplasts
    2.5 Hairy-root transformation
        2.5.1 Agrobacterium culture and infection medium:
        2.5.2 Peanut plants for transformation
        2.5.3 Agrobacterium rhizogenes-mediated transformation
    2.6 Cotyledonary nodes transformation
        2.6.1 Agrobacterium preparation and transformed receptor
        2.6.2 A. tumefaciens-mediated peanut cotyledonary nodes transformation
    2.7 Half-seed transformation
        2.7.1 Explant preparation
        2.7.2 A. tumefaciens-mediated peanut half-seed transformation
    2.8 Identification of mutation in the transformed peanut plants by CRISPR/Cas9 system
        2.8.1 PCR assay for validation of transformation
        2.8.2 PCR-Restriction enzyme （PCR-RE） assay for detection of mutation
        2.8.3 Sequencing
3 Results
    3.1 Protoplast transformation
    3.2 Hairy-root transformation
        3.2.1 PCR-Restriction enzyme （PCR-RE） assay
        3.2.2 Sequencing of transformed hairy roots
    3.3 Cotyledonary nodes transformation
        3.3.1 Tissue culture
        3.3.2 The identification of transformation
    3.4 Half-seed transformation
        3.4.1 Tissue culture
        3.4.2 The identification of transformation
4 Discussion
    4.1 The selection of target site
    4.2 The methods of transformation using CRISPR/Cas9 system
    4.3 The detection methods of mutagenesis caused by CRISPR/Cas9 system
    4.4 The application foreground of CRISPR/Cas9 system
5 Conclusions
References
Appendix
    Appendix 1
    Appendix 2
    Appendix 3
    Appendix 4
    Appendix 5
    Appendix 6
    Appendix 7
    Appendix 8
    Appendix 9
    Appendix 10
    Appendix 11
    Appendix 12
Acknowledgements



本文編號：3067481