实验植物病毒学

Gene Cloning of Cucumber Mosaic Virus and Some Related
Viral Agents
1.1 Introduction
1.2 A Tomato Strain of Cucumber Mosaic Virus， a Natural Reassortant Between Subgroups IA and II
1.3 The Araceae Strain of Cucumber Mosaic Virus Infecting Pinellia ternate Suggested to be a Novel Class Unit Under Subgroup I
1.3.1 Phylogenetice and Sequence Divergence Analysis of 3a and CP ORFs
1.3.2 Phylogenetice and Sequence Divergence Analysis of 5 UTR and 3 UTR， 2a and 2b ORFs of RNA3
1.4 The Potyvirus Infecting Pinellia ternata is a Recombinant Contributed by Soybean Mosaic Virus and Lettuce Mosaic Virus
1.4.1 DAS-ELISA Analysis of Field Samples for Detecting the Potyvirus
1.4.2 Sequencing and Nucleotide Sequence Analysis of the Potyvirus Infecting Pinellia
1.4.3 Amino Acid Sequence Analysis for CP of the Potyvirus Infecting Pinellia
1.4.4 Nucleotide Sequence Analysis for CP N-terminal of the Potyvirus Infecting Pinellia
1.4.5 Amino Acid Sequences for N-terminal and for the Conserved Region of the Potyvirus Infecting Pinellia
1.4.6 Nucleotide Sequences for 3 UTR of the Potyvirus Infecting Pinellia
1.4.7 The General Character and Possible Origin of the Potyvirus Infecting Pinellia
1.5 The 5 Terminal and a Single Nucleotide Determine the Accumulation of Cucumber Mosaic Virus Satellite RNA
1.5.1 GUUU- in 5 Terminal is Necessary to Initiate Replication of 2msatRNA
1．5．2 Typical Structure at the Terminal is Necessary for Long-distance Movement or High？ Accumulation of 2rosatRNK
1．5．3 Low Accumulation of2mF5sat Mutants is Related to Single Nucleotide Mutation
1．5．4 Secondary Structure of2mF5sat Impaired its Replication Capacity
1．6 Methodology
1．6．1 Purification of CMV Virions from Plant Tissue
1．6．2 ImPCR and cDNA Cloning for Full．1ength Genomic RNAs of Cucumber Mosaic Virus
1．6.3 IUl_PCR and Gene Cloning for 3’．end of viral Genome of Soybean Mosaic Virus
1．6．4 Sequence Analysis and Phylogenetice Analysis
1．6．5 Pseudo．recombination of Satellite RNA of Cucumber Mosaic Virhs and the Helper Virus
References
2 Molecular Detection of Cucumber Mosaic VirUS and Other RNA Viruses Based on New Techniques
2．1 Introduction
2．2 Multiplex RT-PCR System for Simultaneous Detection ofFive Potato Viruses
2．2．1 Comparison of 18S rRNA and Had2 mRNA as Internal Controls
2．2．2 The Optimized System for Simultaneous Detection of Potato Viruses with Multiplex ImPCR
2．2．3 Sensitivities of Multiplex RT_PCR and DAS．ELISA in Detecting Potato Viruses
2．3 Detection of Cucumber Mosaic Virus Subgroups and Tobamoviruses Infecting Tomato
2．3．1 Multiplex RT-PCR for Simultaneous Detection of Strains of CMV and TOMV in Tomato
2．3．2 Field Detection of Tomato Viruses by Multiplex RT-PCR
2．3．3 Identification of CMV Subgroups by Restriction Enzymes
uses and Viroids
2．4 A Novel Glass Slide Hybridization for Detecting Plant RNA Viruses and Viroids
2．4．1 Preparation of Highly Sensitive Fluorescent-labeled Probes
2．4．2 Effect of Spotting Solutions on Spot Quality
2．4．3 Effect of Glass Surface Chemistries on Efficiencies of RNA Binding
2.4.4 Detection Limits of Glass Slide Hybridization and Nylon Membrane Hybridization
2.4.5 Specificity of Glass Slide Hybridization
2.4.6 Detection of PVY and PSTVd from Field Potato Samples
2.5 Quantitative Determination of CMV Genome RNAs in Virions by Real-time RT-PCR
2.5.1 Optimization of Real-time RT-PCR and the Specificity
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 Assays
2.6 Accurate and Efficient Data Processing for Quantitative Real-time PCR
2.6.1 Quantification of CMV RNAs in Virions with Standard Curves
2.6.2 Quantification of CMV RNAs in Virions by SCF
2.6.3 Quantification of CMV RNAs in Virions by LinReg PCR and DART Programs
2.6.4 Determination of the Suppression Effect of Satellite RNA on CMV Accumulation in Plant Tissues Using No Values
2.7 Methodology
2.7.1 Primers Design and Specificity Tests in RT-PCR
2.7.2 Comparison of 18S rRNA and nad2 mRNA as Internal Controls
2.7.3 Optimization of Multiplex RT-PCR
2.7.4 Comparison of Sensitivities for Multiplex RT-PCR and DAS-ELISA
2.7.5 Glass Slide Hybridization
References
Infectious Clones and Chimerical Recombination of Cucumber Mosaic
Virus and its Satellite RNAs
3.1 Introduction
3.2 Cucumber Mosaic Virus-mediated Regulation of Disease Development Against Tomato Mosaic Virus in the Tomato
3.2.1 ToMV-N5 Initiated Necrosis on Tomato Can be Protected by Previous Inoculation with Wild-type CMV
3.2.2 ToMV-N5 Initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with CMVA2b
3.2.3 ToMV-N5-initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with Potato Virus X
3.2.4 CMV-initiated Protection against ToMV-N5 is Related to the Replication and Accumulation of Challenging Virus
3.3 Pseudo-recombination between Subgroups of Cucumber Mosaic Virus Demonstrates Different Pathotypes and Satellite RNA Support Characters
3.3.1 Wildtype and Pseudo-recombinants and with or without satRNA Induce Different Symptoms on N. glutinosa
3.3.2 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on N. benthamiana
3.3.3 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on Tomato Varieties
3.3.4 The Pathogenicity of Wildtype and Pseudorecombinants with or without satRNA-Tsh are Related to Viral Accumulation
3.4 Synergy via Cucumber Mosaic Virus and Zucchini Yellow Mosaic
Virus on Cucurbitaceae Hosts
3.4.1 Assessment of Symptom and Synergic Interaction by Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus
3.4.2 Accumulation Kinetics for CMV ORFs in Single or Complex Infection
3.4.3 Accumulation Kinetics ofZYMV CP ORF in Single or Complex Infection
3.5 Methodology
3.5.1 The Interaction Study on CMV and ToMV Interaction
3.5.2 Pseudo-recombination of CMV Subgroups
3.5.3 Synergy between CMV and ZYMV on Cucurbitaceae References
4 Gene Function of Cucumber Mosaic Virus and its Satellite RNA Regarding Viral-host Interactions
4.1 Introduction
4.2 The 2b Protein of Cucumber Mosaic Virus is a Determinant of Pathogenicity and Controls Symptom Expression
4.2.1 Infectivity and Stability of Fny-CMV Derived Mutants
4.2.2 Replacement of the 2b ORF Affected Capsidation of Viral RNA 2
4.2.3 Intraspecies Hybrid Viruses by Changing 2b Gene Induce Different Virulence
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