Development of Wheat Lines Resistant to Fusarium Head Blight
发布时间：2021-04-06 09:48
小麦是人类的主要食物来源之一,中国是最大的小麦生产国。确保小麦的优质和产量,对人类的生存和发展和维护世界和平具有重要意义。然而,小麦生产受到许多因素的影响,尤其是病虫害。由禾谷镰刀菌（Fusarium graminearum,Fg）引起的小麦赤霉病（Fusarim head blight,FHB）,是世界范围内的一种小麦真菌病害,特别是在中国,造成了巨大的经济损失。在禾谷镰刀菌感染植物的过程中,能够产生的多种真菌毒素,这些在真菌毒素主要残留在小麦种子中。真菌毒素严重影响小麦的品质,在摄入百万分之几或百万万分之几微量摄入,对人畜健康构成危险。小麦赤霉病常发生在温暖潮湿的地区,近年来由于全球变暖,FHB呈现流行病害趋势。自2000年以来,在中国的小麦赤霉病经常达到流行病水平。在过去几年中,中国几个小麦主产区频繁的爆发小麦赤霉病的疫情,这越来越引起人们对FHB控制的担忧。在控制赤霉病的手段中,应用杀菌剂成本高昂,而且对环境不友好。选育抗病品种是迄今为止控制真菌病害的最佳技术。抗病育种常用的方法包括常规选择、标记辅助选择（Marker-assisted selection,MAS）和基因工程。...
【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】：133 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
TABLE OF ABBREVIATION
1 GENERAL INTRODUCTION AND LITERATURE REVIEW
1.1 Overview of Wheat
1.2 The Fungus Fusarium graminearum
1.2.1 Taxonomy,Pathology,and Ecology
1.2.2 Parasitic Life Cycle
1.2.3 Formation of Mycotoxins
1.2.4 Symptoms of F.graminearum on Cereals
1.3 Management of FHB and RNAi
1.3.1 RNAi Application in Plants against Fungi
1.3.2 Basic Mechanism of RNAi
1.3.3 Components of RNAi Machinery
1.3.4 Different Methods to Trigger RNAi
1.3.4.1 Host-induced gene silencing（HIGS）
1.3.4.2 Hairpin RNAi（hp RNAi）
1.3.4.3 Virus-induced gene silencing（VIGS）
1.3.4.4 mi RNA induced gene silencing（MIGS）
1.3.4.5 Using artificial mi RNAs（ami RNAs）
1.3.4.6 VIGS using ami RNAs（MIR-VIGS）
1.3.4.7 Spray induced gene silencing（SIGS）
1.3.4.8DNAi
1.3.5 Critical Factors for Successful RNAi Induction
1.4 Protein Kinase C（Pk C）
1.5 Chitin Synthase7 （Chs7）
1.6 Aim of this Study
2 MATERIALS AND METHODS
2.1 Experimental Materials
2.1.1 Plant Material
2.1.2 Fungal Strain
2.1.3 Enzymes,Reagents,and Kits
2.1.4 Reagents and Culture Media
2.1.4.1 DNA extraction reagents by CTAB method
2.1.4.2 Northern blot solutions
2.2 Test Methods
2.2.1 Sequence Analysis and3D Structure Prediction
2.2.2 RNA Extraction by TRIzol
2.2.2.1 Preparation of samples
2.2.2.2 RNA extraction
2.2.3 First Strand c DNA Synthesis
2.2.4 PCR Amplification（Easy Taq Enzyme）
2.2.5 Wheat Genomic DNA Extraction by CTAB Method
2.2.6 Fusarium Head Blight Assay
2.2.6.1 Preparation of fungal spore suspension
2.2.6.2 Counting of spores by hemocytometers
2.2.6.3 Fusarium Seedling blight Assay
2.2.6.4 Single flower inoculation
2.2.7 Powdery Mildew Assay
2.2.8 Northern Blot
2.2.8.1 Sample Preparation
2.2.8.2 Gel preparation and electrophoresis
2.2.8.3 Transfer
2.2.8.4 Prehybridization
2.2.8.5 Hybridization
2.2.8.6 Washing and exposing
2.2.9 Quantitative RT-PCR Protocol（q RT-PCR）
2.2.9.1 Primer design criteria
2.2.9.2 Method
2.2.10 Mycotoxin Determination by Gass Chromatography-Mass Spectrometry
2.2.11 Evaluation of Agronomic Traits
2.2.12 Statistical Analysis
3 RESULTS
3.1 Identification of Essential Genes
3.2 Prediction of Protein Structures
3.3 Prediction of Conserved Domain
3.4 Multiple Sequence Alignment
3.5 Construction of Phylogenetic Trees
3.6 Identification of Gene-Specific Sequence Tags（GSTs）
3.7 Prediction of Off-Targets
3.8 Generation of Transgenic Lines
3.8.1 Transgenic lines developed via biolistic transformation
3.8.2 Transgenic lines developed via Agrobacterium mediated transformation
3.9 Establishing Homozygosity and Screening of Transgenic Lines
3.10 Evaluation of Transgenic Lines against Fusarium Seedling Blight
3.11 Evaluation of Transgenic Lines against Fusarium Head Blight
3.12 Quantification of Fungal Transcripts Levels in Transgenic Lines
3.13 Detection of si RNAs in Transgenic Lines
3.14 Evaluation of Mycotoxin Resistance in Transgenic Lines
3.15 Evaluation of Transgenic Lines against Powdery Mildew Disease
3.16 Evaluation of Agronomic Traits in Transgenic Lines
4 DISCUSSION
4.1 Selection of Essential Genes
4.2 Sequence Analysis of Genes
4.3 Factors Affecting RNAi Efficiency
4.4 Factors Affecting Transformation Efficiency
4.5 Evaluation of Transgenic Lines
4.6 Future Prospects
REFERENCES
Acknowledgment
本文编号：3121228
【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】：133 页
【学位级别】：博士
【文章目录】：
ABSTRACT
摘要
TABLE OF ABBREVIATION
1 GENERAL INTRODUCTION AND LITERATURE REVIEW
1.1 Overview of Wheat
1.2 The Fungus Fusarium graminearum
1.2.1 Taxonomy,Pathology,and Ecology
1.2.2 Parasitic Life Cycle
1.2.3 Formation of Mycotoxins
1.2.4 Symptoms of F.graminearum on Cereals
1.3 Management of FHB and RNAi
1.3.1 RNAi Application in Plants against Fungi
1.3.2 Basic Mechanism of RNAi
1.3.3 Components of RNAi Machinery
1.3.4 Different Methods to Trigger RNAi
1.3.4.1 Host-induced gene silencing（HIGS）
1.3.4.2 Hairpin RNAi（hp RNAi）
1.3.4.3 Virus-induced gene silencing（VIGS）
1.3.4.4 mi RNA induced gene silencing（MIGS）
1.3.4.5 Using artificial mi RNAs（ami RNAs）
1.3.4.6 VIGS using ami RNAs（MIR-VIGS）
1.3.4.7 Spray induced gene silencing（SIGS）
1.3.4.8DNAi
1.3.5 Critical Factors for Successful RNAi Induction
1.4 Protein Kinase C（Pk C）
1.5 Chitin Synthase7 （Chs7）
1.6 Aim of this Study
2 MATERIALS AND METHODS
2.1 Experimental Materials
2.1.1 Plant Material
2.1.2 Fungal Strain
2.1.3 Enzymes,Reagents,and Kits
2.1.4 Reagents and Culture Media
2.1.4.1 DNA extraction reagents by CTAB method
2.1.4.2 Northern blot solutions
2.2 Test Methods
2.2.1 Sequence Analysis and3D Structure Prediction
2.2.2 RNA Extraction by TRIzol
2.2.2.1 Preparation of samples
2.2.2.2 RNA extraction
2.2.3 First Strand c DNA Synthesis
2.2.4 PCR Amplification（Easy Taq Enzyme）
2.2.5 Wheat Genomic DNA Extraction by CTAB Method
2.2.6 Fusarium Head Blight Assay
2.2.6.1 Preparation of fungal spore suspension
2.2.6.2 Counting of spores by hemocytometers
2.2.6.3 Fusarium Seedling blight Assay
2.2.6.4 Single flower inoculation
2.2.7 Powdery Mildew Assay
2.2.8 Northern Blot
2.2.8.1 Sample Preparation
2.2.8.2 Gel preparation and electrophoresis
2.2.8.3 Transfer
2.2.8.4 Prehybridization
2.2.8.5 Hybridization
2.2.8.6 Washing and exposing
2.2.9 Quantitative RT-PCR Protocol（q RT-PCR）
2.2.9.1 Primer design criteria
2.2.9.2 Method
2.2.10 Mycotoxin Determination by Gass Chromatography-Mass Spectrometry
2.2.11 Evaluation of Agronomic Traits
2.2.12 Statistical Analysis
3 RESULTS
3.1 Identification of Essential Genes
3.2 Prediction of Protein Structures
3.3 Prediction of Conserved Domain
3.4 Multiple Sequence Alignment
3.5 Construction of Phylogenetic Trees
3.6 Identification of Gene-Specific Sequence Tags（GSTs）
3.7 Prediction of Off-Targets
3.8 Generation of Transgenic Lines
3.8.1 Transgenic lines developed via biolistic transformation
3.8.2 Transgenic lines developed via Agrobacterium mediated transformation
3.9 Establishing Homozygosity and Screening of Transgenic Lines
3.10 Evaluation of Transgenic Lines against Fusarium Seedling Blight
3.11 Evaluation of Transgenic Lines against Fusarium Head Blight
3.12 Quantification of Fungal Transcripts Levels in Transgenic Lines
3.13 Detection of si RNAs in Transgenic Lines
3.14 Evaluation of Mycotoxin Resistance in Transgenic Lines
3.15 Evaluation of Transgenic Lines against Powdery Mildew Disease
3.16 Evaluation of Agronomic Traits in Transgenic Lines
4 DISCUSSION
4.1 Selection of Essential Genes
4.2 Sequence Analysis of Genes
4.3 Factors Affecting RNAi Efficiency
4.4 Factors Affecting Transformation Efficiency
4.5 Evaluation of Transgenic Lines
4.6 Future Prospects
REFERENCES
Acknowledgment
本文编号：3121228
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