实验植物病毒学 英文版PDF|Epub|txt|kindle电子书版本网盘下载

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1 Gene Cloning of Cucumber Mosaic Virus and Some Related Viral Agents1

1.1 Introduction1

1.2 A Tomato Strain of Cucumber Mosaic Virus,a Natural Reassortant Between Subgroups IA and Ⅱ5

1.3　The Araceae Strain of Cucumber Mosaic Virus Infecting Pinellia ternate Suggested to be a Novel Class Unit Under Subgroup Ⅰ10

1.3.1　Phylogenetice and Sequence Divergence Analysis of 3a and CP ORFs15

1.3.2　Phylogenetice and Sequence Divergence Analysis of 5′ UTR and 3′UTR,2a and 2b ORFs of RNA315

1.4　The Potyvirus Infecting Pinellia ternata is a Recombinant Contributed by Soybean Mosaic Virus and Lettuce Mosaic Virus17

1.4.1 DAS-ELISA Analysis of Field Samples for Detecting the Potyvirus18

1.4.2　Sequencing and Nucleotide Sequence Analysis of the Potyvirus Infecting Pinellia20

1.4.3 Amino Acid Sequence Analysis for CP of the Potyvirus Infecting Pinellia22

1.4.4 Nucleotide Sequence Analysis for CP N-terminal of the Potyvirus Infecting Pinellia24

1.4.5 Amino Acid Sequences for N-terminal and for the Conserved Region of the Potyvirus Infecting Pinellia25

1.4.6 Nucleotide Sequences for 3′UTR of the Potyvirus Infecting Pinellia27

1.4.7　The General Character and Possible Origin of the Potyvirus Infecting Pinellia27

1.5 The 5′Terminal and a Single Nucleotide Determine the Accumulation of Cucumber Mosaic Virus Satellite RNA31

1.5.1 GUUU-in 5′Terminal is Necessary to Initiate Replication of 2msatRNA32

1.5.2　Typical Structure at the 5′Terminal is Necessary for Long-distance Movement or High Accumulation of 2msatRNA34

1.5.3　Low Accumulation of 2mF5sat Mutants is Related to Single Nucleotide Mutation36

1.5.4 Secondary Structure of 2mF5sat Impaired its Replication Capacity38

1.6　Methodology40

1.6.1 Purification of CMV Virions from Plant Tissue40

1.6.2　RT-PCR and cDNA Cloning for Full-length Genomic RNAs of Cucumber Mosaic Virus41

1.6.3　RT-PCR and Gene Cloning for 3′-end of Viral Genome of Soybean Mosaic Virus42

1.6.4　Sequence Analysis and Phylogenetice Analysis43

1.6.5　Pseudo-recombination of Satellite RNA of Cucumber Mosaic Virus and the Helper Virus43

References44

2 Molecular Detection of Cucumber Mosaic Virus and Other RNA Viruses Based on New Techniques47

2.1 Introduction47

2.2 Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses51

2.2.1 Comparison of 18S rRNA and nad2 mRNA as Internal Controls52

2.2.2　The Optimized System for Simultaneous Detection of Potato Viruses with Multiplex RT-PCR54

2.2.3 Sensitivities of Multiplex RT-PCR and DAS-ELISA in Detecting Potato Viruses55

2.3　Detection of Cucumber Mosaic Virus Subgroups and Tobamoviruses Infecting Tomato57

2.3.1 Multiplex RT-PCR for Simultaneous Detection of Strains of CMV and ToMV in Tomato58

2.3.2 Field Detection of Tomato Viruses by Multiplex RT-PCR62

2.3.3 Identification of CMV Subgroups by Restriction Enzymes62

2.4 A Novel Glass Slide Hybridization for Detecting Plant RNA Viruses and Viroids65

2.4.1　Preparation of Highly Sensitive Fluorescent-labeled Probes66

2.4.2　Effect of Spotting Solutions on Spot Quality67

2.4.3 Effect of Glass Surface Chemistries on Efficiencies of RNA Binding68

2.4.4　Detection Limits of Glass Slide Hybridization and Nylon Membrane Hybridization69

2.4.5 Specificity of Glass Slide Hybridization71

2.4.6　Detection of PVY and PSTVd from Field Potato Samples72

2.5 Quantitative Determination of CMV Genome RNAs in Virions by Real-time RT-PCR74

2.5.1 Optimization of Real-time RT-PCR and the Specificity76

2.5.2 Quantification of CMV Genomic RNAs by RT-PCR and Comparison of the Quantification with Lab-on-a-Chip and Northern Blot Hybridization Assays76

2.6 Accurate and Efficient Data Processing for Quantitative Real-time PCR81

2.6.1 Quantification of CMV RNAs in Virions with Standard Curves82

2.6.2 Quantification of CMV RNAs in Virions by SCF83

2.6.3 Quantification of CMV RNAs in Virions by LinReg PCR and DART Programs85

2.6.4　Determination of the Suppression Effect of Satellite RNA on CMV Accumulation in Plant Tissues Using N0 Values87

2.7　Methodology88

2.7.1　Primers Design and Specificity Tests in RT-PCR88

2.7.2 Comparison of 18S rRNA and nad2 mRNA as Internal Controls89

2.7.3 Optimization of Multiplex RT-PCR90

2.7.4 Comparison of Sensitivities for Multiplex RT-PCR and DAS-ELISA91

2.7.5 Glass Slide Hybridization91

References94

3 Infectious Clones and Chimerical Recombination of Cucumber Mosaic Virus and its Satellite RNAs97

3.1 Introduction97

3.2 Cucumber Mosaic Virus-mediated Regulation of Disease Development Against Tomato Mosaic Virus in the Tomato98

3.2.1　ToMV-N5 Initiated Necrosis on Tomato Can be Protected by Previous Inoculation with Wild-type CMV100

3.2.2　ToMV-N5 Initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with CMV△2b102

3.2.3 ToMV-N5-initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with Potato Virus X103

3.2.4 CMV-initiated Protection against ToMV-N5 is Related to the Replication and Accumulation of Challenging Virus104

3.3　Pseudo-recombination between Subgroups of Cucumber Mosaic Virus Demonstrates Different Pathotypes and Satellite RNA Support Characters105

3.3.1 Wildtype and Pseudo-recombinants and with or without satRNA Induce Different Symptoms on N.glutinosa105

3.3.2 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on N.benthamiana107

3.3.3 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on Tomato Varieties108

3.3.4　The Pathogenicity of Wildtype and Pseudorecombinants with or without satRNA-Tsh are Related to Viral Accumulation110

3.4 Synergy via Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus on Cucurbitaceae Hosts111

3.4.1 Assessment of Symptom and Synergic Interaction by Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus112

3.4.2 Accumulation Kinetics for CMV ORFs in Single or Complex Infection113

3.4.3 Accumulation Kinetics of ZYMV CP ORF in Single or Complex Infection116

3.5　Methodology117

3.5.1 The Interaction Study on CMV and ToMV Interaction118

3.5.2　Pseudo-recombination of CMV Subgroups119

3.5.3　Synergy between CMV and ZYMV on Cucurbitaceae121

References122

4　Gene Function of Cucumber Mosaic Virus and its Satellite RNA Regarding Viral-host Interactions125

4.1 Introduction125

4.2　The 2b Protein of Cucumber Mosaic Virus is a Determinant of Pathogenicity and Controls Symptom Expression127

4.2.1 Infectivity and Stability of Fny-CMV Derived Mutants128

4.2.2 Replacement of the 2b ORF Affected Capsidation of Viral RNA 2130

4.2.3　Intraspecies Hybrid Viruses by Changing 2b Gene Induce Different Virulence132

4.2.4　Divertive Virulence is Mediated by the 2b Protein Rather than by the C-terminal Overlapping Parts of the 2a Protein132

4.2.5 Virulence is Associated with the Accumulation of Viral Progeny RNAs Affected by 2b Protein134

4.3 Function of CMV 2b Protein and the C-terminus of 2a Protein in Determining Viral RNA Accumulation and Symptom Development137

4.3.1 The Systemic Necrosis-inducing Domain is Related to a 125-nucleotide Region of RNA 2138

4.3.2 Effect of 2b Protein Amino Acid 55 on Viral Accumulation and Symptom Development140

4.3.3 Sequence Analyses of the 2b Proteins and the C-top of the 2a Proteins141

4.3.4 Effect of the C-terminus of 2a Protein on Symptom Expression and Virus Accumulation143

4.4 Satellite RNA-mediated Reduction in Accumulation of CMV Genomic RNAs in Tobacco Related to 2b Gene of the Helper Virus146

4.4.1 Symptom Expression on N.Tabacum Inoculated with CMV-Fsat146

4.4.2 Effect of satRs on the Accumulation of CMV-Fny Genomic RNAs148

4.4.3 Symptom Expression on the Host Plants Inoculated with CMV-Fny△2b148

4.4.4 Accumulation of CMV-Fny△2b Genomic RNAs and the Effect of satRNAs149

4.4.5 Accumulation of CMV-Fny Genomic RNAs in the Inoculated Leaves and the Effect of satRNAs151

4.4.6　The Effect of satRNAs on Long-distance Movement of CMV-Fny Genomic RNAs152

4.5　Methodology153

4.5.1　Plants,Viruses and Plasmid Constructs153

4.5.2　Plant Inoculation and Viral Progeny RNA Analysis158

4.5.3　Quantifying the Accumulation of Viral RNAs in Leaf Tissue159

References159

5　Plant MicroRNAs and Their Response to Infection of Plant Viruses163

5.1 Introduction163

5.2　Methodology165

5.2.1 Computational Prediction of miRNAs and Their Target Genes for Plant Species with Known Genome Sequences165

5.2.2 Use Plant miRNA Microwarrays to Identify Conservative miRNAs from New Host Plants167

5.2.3 Use Plant miRNA Microarrays to Identify Conservative miRNAs Response to Virus Infection169

5.2.4 Quantitative Determination of miRNAs by Stem-loop Real-time RT-PCR170

5.2.5　Design of Plant miRNA-array and Data Analysis173

5.2.6 Confirmation of miRNAs by Northern Blotting and Target mRNA by 3′-RACE174

5.3　Tomato miRNAs Predicted from Known Genomic Sequences and Discovered by miRNA Microarray174

5.3.1　Potential Tomato miRNAs Predicted Computationally According to Known Genomic Sequences175

5.3.2　Potential Targets of Newly Predicted miRNAs and Their Function178

5.3.3 Confirmation of Tomato miRNAs Expression and Survey by Microarray180

5.4 Mechanisms Involved in Plant miRNA Expression with Regard to Infection of ssRNA Viruses185

5.4.1　Phenotype in Tomato Under Infection with CMV/satRNA Combinations and ToMV186

5.4.2 Response of Tomato miRNA Expression to Virus Infection187

5.4.3 MiRNA Expression Profiles between CMV-Fny and CMV-Fny△2b Infections193

5.4.4 MiRNAs Expression Profiles Altered with Addition of satRNAs194

5.4.5 A Comparison of miRNAs Expression Profiles between CMV and ToMV Infections195

5.5　Tomato miRNA Response to Virus Infection Quantified by Real-time RT-PCR197

5.5.1 Identification of Tomato ARF8-and AGO1-like Genes199

5.5.2 Analytical Validation of Real-time RT-PCR for Amplification of miRNAs200

5.5.3　Quantification of Tomato miRNAs Expression by Stem-loop Real-time RT-PCR202

5.5.4 Quantification of miRNAs Targets in Tomato under Cucumovirus Infection204

References206

6 Genomic Characterization of New Viruses with Double Stranded RNA Genomes211

6.1 Introduction211

6.2 Novel dsRNA Viruses Infecting Raphanus sativus212

6.2.1　Yellow Edge Symptoms and dsRNA Patterns in the Radish213

6.2.2 Genome Characterization of Raphanus sativus Cryptic Virus 1217

6.2.3 Genome Characterization of Raphanus sativus Cryptic Virus 2222

6.2.4 Correlation of Raphanus sativus Cryptic Virus 2 with Raphanus sativus Cryptic Virus 1224

6.2.5 Genome Characterization of Suggested Raphanus sativus Cryptic Virus 3226

6.2.6　The Possible Existence of More dsRNA Viruses in Radish229

6.3　Double Stranded Virnses in Vicia faba229

6.3.1　Two dsRNA Viruses Infecting V.faba230

6.3.2　A Partitiviruss Infecting Aspergilus sp.Associated with Leaf Tissue of Vicia faba237

6.4 A Novel dsRNA Virus Infecting Primula malacoides Franch243

6.5　Derivation and Evolutionary Relationship of dsRNA Viruses Infecting plants249

6.6　Conclusion257

6.7　Methodology258

6.7.1　Plant Material and dsRNA Extraction258

6.7.2　Purification of Virus Particles260

6.7.3　Amplification of Unknown dsRNA Sequence by Modified Single-primer Amplification Technique(SPAT)260

6.7.4　Sequence Analysis261

6.7.5 Dot-Blot Hybridization262

References262

Index267