水稻镉运输基因OsLCT1和OsNramp5的特征与突变分析
发布时间：2021-08-20 06:27
水稻是重要的粮食作物之一,全国有60%人口以稻米为主食,随着人们生活水平的提高,对稻米品质要求日渐提高。重金属镉（Cd）是一种非必需元素,对动植物生长发育有毒害作用。工业废水污染、矿产废弃物排放、以及矿质肥料的使用,造成了一些农田Cd含量超标,进而造成稻米Cd超标,严重危害人民健康。为此,国家已采用多种土壤修复措施降低土壤Cd污染的影响,但进展较为缓慢。为实现镉污染土壤中的水稻安全生产,本研究采用CRISPR/Cas9基因组编辑技术创建了OsLCT1和OsNramp5的水稻突变株系,以期阻止Cd的吸收和转运。在此基础上,还对其中两个OsLCT1突变体（lct1x1和lct1x3）和两个OsNramp5突变体（nramp5×7和nramp5×9）,通过盆栽和田间试验,进行了稻谷镉积累和农艺性状评估,对四个OsNramp5突变体（nramp5×7+1,nramp5×7-2,nramp5×9+1和nramp5×9-33）通过水培进行了生理性状和转录组分析。主要结果总结如下:1.利用CRSPR/Cas9技术快速创建了多种类型的OsLCT1和OsNramp5水稻突变株系。利用本实验室改良的CRI...
【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J]. 邵高能,谢黎虹,焦桂爱,魏祥进,圣忠华,唐绍清,胡培松. 中国水稻科学. 2017(02)
本文编号：3352998
【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
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
【参考文献】：
期刊论文
[1]利用CRISPR/CAS9技术编辑水稻香味基因Badh2[J]. 邵高能,谢黎虹,焦桂爱,魏祥进,圣忠华,唐绍清,胡培松. 中国水稻科学. 2017(02)
本文编号：3352998
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