利用CRISPR/Cas9编辑水稻木质素单体合成途径基因
发布时间：2021-10-15 16:23
在维管植物中,木质素是次生细胞壁中含量仅次于纤维素的生物大分子,在植物应力响应、机械支持和水分传输中起着重要作用。木质素生物合成相关基因的表达、演化和功能研究为研究植物次生代谢通路的协调进化提供了一个极好的模型,同时对秸秆的理化性质的遗传改良具有重要的意义。CRISPR/Cas9基因组编辑技术因其兼具高效、精准、易用性和通用性等优点。本研究分析了水稻木质素单体合成途径相关基因,利用CREP数据库发现了27个表达量较高的基因,从中选取了肉桂醇脱氢酶基因CAD1、肉桂酸4-羟化酶基因C4H2、羟基肉桂酰转移酶HCT1和HCT2这4个基因作为本研究的对象。利用CRISPR/Cas9系统进行了编辑,目前已经通过农杆菌介导的成功获得了相关的转基因阳性单株,并对具体的编辑位点进行了测序确认,获得了目标基因编辑的材料,为研究和改良木质素单体合成途径建立了基础。同时,本研究还对这些基因编码的CAD1、C4H2、HCT1和HCT2蛋白质的3 D结构,以及禾本科植物中的同源蛋白进行了比较研究和聚类分析。该研究有望通过木质素单体H、S和G配比的改造,为研究水稻植株的农艺适应性、秸秆的理化性质的改变和秸秆的综...
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：105 页
【学位级别】：硕士
【文章目录】：
摘要
ABSTRACT
ABBREVIATION
1 INTRODUCTION
1.1 Literature review
1.1.1 Rice as a Model Plant
1.1.2 Lignin and Lignocellulosic Biomass Conversion
1.1.3 The Lignin Monolignol Biosynthetic Pathway
1.1.4 The Recent Developments in Transgenic Technologies and CRISPR/Cas9 Gene-Editing System
1.2 Research Objectives
2 MATERIAL AND METHOD
2.1 Selection of Genes
2.2 Designing sgRNA
2.3 Vector Construction
2.3.1 Primer Renaturation
2.3.2 CRISPR Vector Restriction Digestion
2.3.3 DNA and Vector Ligation
2.4 Transform Escherichia coli DH5α Competent Cells
2.4.1 Preparation of DH5α Competent Cells
2.4.2 Transforming DH5α Competent Cells
2.4.3 LB-Medium
2.5 Selection of Positive Colonies
2.5.1 PCR Protocol
2.5.2 Sanger Sequencing
2.6 Sub-Clone Vector into Agrobacterium
2.6.1 Freeze–Thaw Method
2.7 Rice Transformation
2.7.1 Induction of Calli
2.7.2 Preparation of Calli and Agrobacterium
2.7.3 Infection of Callus with Agrobacterium
2.8 Detection of Positive Plants
2.8.1 DNA Extraction
2.8.2 CTAB Extraction Buffer
2.9 Software and Websites Used
3 RESULTS AND ANALYSIS
3.1 Collection of Protein Sequences and Alignment
3.2 Identities of Genes with their Homologous Proteins
3.3 Multiple Sequence Alignment and Phylogenetic Tree
3.4 3D Proteins Structures Prediction
3.5 Identification of Unique Sites in Proteins to Design sgRNA
3.6 Designed sgRNA
3.7 Vector Construction
3.8 Identification of Positive E. coli Colonies and Sequencing Results
3.9 Sub-Cloning of Plasmid into Agrobacterium tumefaciens
3.10 T0 Generation Mutants
3.11 Mutations Observed in T0 Generation
3.12 Phenotypes Observed in Mutant Plants
3.13 Mutants Protein Sequence Predictions and Alignment
4 DISCUSSION
4.1 Some Remaining Issues
REFERENCES
Acknowledgements
【参考文献】：
期刊论文
[1]Genetic Engineering of Energy Crops: A Strategy for Biofuel Production in China[J]. Guosheng Xie1 and Liangcai Peng1,2 1National Key Laboratory of Crop Genetic Improvement,Biomass and Bioenergy Research Centre,and College of Plant Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China 2College of Life Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China. Journal of Integrative Plant Biology. 2011(02)
本文编号：3438273
【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校
【文章页数】：105 页
【学位级别】：硕士
【文章目录】：
摘要
ABSTRACT
ABBREVIATION
1 INTRODUCTION
1.1 Literature review
1.1.1 Rice as a Model Plant
1.1.2 Lignin and Lignocellulosic Biomass Conversion
1.1.3 The Lignin Monolignol Biosynthetic Pathway
1.1.4 The Recent Developments in Transgenic Technologies and CRISPR/Cas9 Gene-Editing System
1.2 Research Objectives
2 MATERIAL AND METHOD
2.1 Selection of Genes
2.2 Designing sgRNA
2.3 Vector Construction
2.3.1 Primer Renaturation
2.3.2 CRISPR Vector Restriction Digestion
2.3.3 DNA and Vector Ligation
2.4 Transform Escherichia coli DH5α Competent Cells
2.4.1 Preparation of DH5α Competent Cells
2.4.2 Transforming DH5α Competent Cells
2.4.3 LB-Medium
2.5 Selection of Positive Colonies
2.5.1 PCR Protocol
2.5.2 Sanger Sequencing
2.6 Sub-Clone Vector into Agrobacterium
2.6.1 Freeze–Thaw Method
2.7 Rice Transformation
2.7.1 Induction of Calli
2.7.2 Preparation of Calli and Agrobacterium
2.7.3 Infection of Callus with Agrobacterium
2.8 Detection of Positive Plants
2.8.1 DNA Extraction
2.8.2 CTAB Extraction Buffer
2.9 Software and Websites Used
3 RESULTS AND ANALYSIS
3.1 Collection of Protein Sequences and Alignment
3.2 Identities of Genes with their Homologous Proteins
3.3 Multiple Sequence Alignment and Phylogenetic Tree
3.4 3D Proteins Structures Prediction
3.5 Identification of Unique Sites in Proteins to Design sgRNA
3.6 Designed sgRNA
3.7 Vector Construction
3.8 Identification of Positive E. coli Colonies and Sequencing Results
3.9 Sub-Cloning of Plasmid into Agrobacterium tumefaciens
3.10 T0 Generation Mutants
3.11 Mutations Observed in T0 Generation
3.12 Phenotypes Observed in Mutant Plants
3.13 Mutants Protein Sequence Predictions and Alignment
4 DISCUSSION
4.1 Some Remaining Issues
REFERENCES
Acknowledgements
【参考文献】：
期刊论文
[1]Genetic Engineering of Energy Crops: A Strategy for Biofuel Production in China[J]. Guosheng Xie1 and Liangcai Peng1,2 1National Key Laboratory of Crop Genetic Improvement,Biomass and Bioenergy Research Centre,and College of Plant Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China 2College of Life Sciences and Technology,Huazhong Agricultural University,Wuhan 430070,China. Journal of Integrative Plant Biology. 2011(02)
本文编号：3438273
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