水稻是重要的糧食作物之一,全國有60%人口以稻米為主食,隨著人們生活水平的提高,對稻米品質(zhì)要求日漸提高。重金屬鎘（Cd）是一種非必需元素,對動植物生長發(fā)育有毒害作用。工業(yè)廢水污染、礦產(chǎn)廢棄物排放、以及礦質(zhì)肥料的使用,造成了一些農(nóng)田Cd含量超標,進而造成稻米Cd超標,嚴重危害人民健康。為此,國家已采用多種土壤修復措施降低土壤Cd污染的影響,但進展較為緩慢。為實現(xiàn)鎘污染土壤中的水稻安全生產(chǎn),本研究采用CRISPR/Cas9基因組編輯技術(shù)創(chuàng)建了OsLCT1和OsNramp5的水稻突變株系,以期阻止Cd的吸收和轉(zhuǎn)運。在此基礎(chǔ)上,還對其中兩個OsLCT1突變體（lct1x1和lct1x3）和兩個OsNramp5突變體（nramp5×7和nramp5×9）,通過盆栽和田間試驗,進行了稻谷鎘積累和農(nóng)藝性狀評估,對四個OsNramp5突變體（nramp5×7+1,nramp5×7-2,nramp5×9+1和nramp5×9-33）通過水培進行了生理性狀和轉(zhuǎn)錄組分析。主要結(jié)果總結(jié)如下:1.利用CRSPR/Cas9技術(shù)快速創(chuàng)建了多種類型的OsLCT1和OsNramp5水稻突變株系。利用本實驗室改良的CRI... 

【文章來源】：浙江大學浙江省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：131 頁

【學位級別】：博士

【文章目錄】：
Acknowledgements
摘要
Abstract
Abbreviation
Chapter1.Literature Review
    1.1 Pollution and Transport of Cd
        1.1.1 Cd Pollution
        1.1.2 The Mechanism of Cd Transport in Plants
        1.1.3 Cd Stress
    1.2 Effect and Transport of Mn
        1.2.1 Mn in Paddy Field
        1.2.2 The Mechanism of Mn Transport in Plants
        1.2.3 Mn Stress
            1.2.3.1 Effects and Mechanisms of Mn Limitation
            1.2.3.2 Effects and Mechanisms of Mn Toxicity
    1.3 Genome Editing using CRISPR/Cas9 System
        1.3.1 Defense System Pathway of CRISPR/Cas System
        1.3.2 Cas9 Nuclease Activity
    1.4 Objectives and Contents of This Study
Chapter2 Targeted Mutagenesis of Genes involved in Cadmium Transport
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Target Site Selection
        2.2.2 CRISPR Vector Construction
        2.2.3 Transformation into E.coli
        2.2.4 Transformation into Agrobacterium
        2.2.5 Agrobacterium-mediated Rice Transformation
        2.2.6 Transgene and Mutation Identification
    2.3 Results
        2.3.1 Generation of OsLCT1 Mutants
            2.3.1.1 Gene Editing Vectors Constructed for OsLCT1
            2.3.1.2 Generation of Transgenic Lines
            2.3.1.3 Mutant T0 Plants Revealed by HRM Analysis
            2.3.1.4 Mutant T0 Plants Checked by Sequencing
            2.3.1.5 Development of Oslct1 Transgene-free Mutant Lines
        2.3.2 Generation of OsNramp5 Mutants
            2.3.2.1 Gene Editing Vectors Constructed for OsNramp
            2.3.2.2 Generation of Transgenic Lines of osnramp
            2.3.2.3 Mutant T_0 Plants Revealed by HRM Analysis
            2.3.2.4 Mutations in T_0 Plants Confirmed by Sequencing
            2.3.2.5 Development of osnramp5 Transgene-free Mutant Lines
        2.3.3 Development of Homozygous Transgene-free Mutant Lines of oslct1 and osnramp
    2.4 Discussion
Chapter3.Effect of oslct1 and osnramp5 Mutations on Growth and Metal Content
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Pot Experiment
        3.2.2 Paddy Field Experiment
        3.2.3 Digestion and Measurement of Cd in Soil Samples
        3.2.4 Digestion and Measurement of Cd and Mineral Elements in Brown Rice Grains
        3.2.5 Assessment of Agronomic Trait
    3.3 Results
        3.3.1 Effect of oslct1 Mutations on Growth and Metal Content
            3.3.1.1 Effect of oslct1 Mutations on Cd Content in Brown Rice Grains
            3.3.1.2 Effects of oslct1 Mutations on Mineral Accumulation in Rice Grains
            3.3.1.3 Impact of oslct1 Mutations on Plant Growth and Yield
        3.3.2 Effect of osnramp5 Mutations on Growth Grain and Mineral Content
            3.3.2.1 Effect of osnramp5 Mutations on Cd Content in Brown Rice Grains
            3.3.2.2 Effects of osnramp5 Mutations on Grain Mineral Accumulation in Rice Grain
            3.3.2.3 Impact of osnramp5 Mutations on Plant Growth and Yield
    3.4 Discussion
        3.4.1 Mutational Effect of Os Nramp5 and Os LCT
        3.4.2 Usefulness of nrmap5x7 and lct1x1 for Rice Production in Cd-polluted Paddy Fields
        3.4.3 Perspectives of Breeding Low-Cd-Accumulating Rice by CRISPR/Cas9-Mediated Mutagenesis of Cd Transporters
Chapter4.Physiological Characteristics and Transcriptome Analysis of osnramp5 Mutants and Wild Type Xidao
    4.1 Introduction
    4.2 Materials and Methods
        4.2.1 Hydroponic Experiments
        4.2.2 Element Extraction in the Heating Block
        4.2.3 Chlorophyll Concentration Measurement
        4.2.4 Preparation of Materials for Transcriptome Sequencing
        4.2.5 RNA Qualification and Quantification
        4.2.6 Preparation of Libraries for Transcriptome Sequencing
    4.3 Results
        4.3.1 Effect of osnramp5 Mutations on Plant Growth
        4.3.2 Effect of osnramp5 Mutations on Element Content in Rice Tissues
        4.3.3 Impact of osnramp5 Mutation and Treatment with Different Mn and Cd Concentration on Chlorophyll Content
        4.3.4 Transcriptome Analysis of Mutants and Wild Type
    4.4 Discussion
        4.4.1 Disturbed OsNramp5 Protein Structure Impact Mn and Cd Transport in Rice Tissues
        4.4.2 The Relationship between Mn and Cd Transport were Different in Root and Shoot
        4.4.3 The osnramp5 Mutations Plays Different Roles in the Six Elements Transport
        4.4.4 Different Phenotypes of Four osnramp5 Mutant Lines
Chapter5.Overall Conclusion and Future Perspectives
Reference


【參考文獻】：
期刊論文
[1]利用CRISPR/CAS9技術(shù)編輯水稻香味基因Badh2[J]. 邵高能,謝黎虹,焦桂愛,魏祥進,圣忠華,唐紹清,胡培松.  中國水稻科學. 2017(02)



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