声明
摘要
Abstract
Table of Contents
List of tables
List of figures
LIST OF ABBREVIATIONS
CHAPTER ONE
1．2 Economic importance of lily viruses
1．3 Potexvirus
1．3．1 Introduction
1．3．2 Plantago asiatica mosaic virus(PlAMV)
1．3．3 Morphology of Potexvirus and PIAMV
1．3．5 Host range and Transmission of PlAMV
1．3．6 Symptoms and Geographical Distribution of PIAMV
1．4 Molecular techniques used for detection of plant viruses
1．4．1 Reverse Transeription Polymerase chain reaction (RT-PCR)
1．4．2 Real-Time Reverse Transcription Polymerase Chain Reaction(RT-qPCR)
1．4．3 Next generation sequencing (NGS)technology or RNA Sequencing(RNA-seq)
1．5 Control measures of lily viruses
1．5．1 Producing virus-free lilies or bulbs
1．5．2 Insect vector control
1．6 The current study
1．6．1 Objectives of this study were
CHAPTER TWO Identification and Molecular Characterization of Plantago asiatica mosaic virus(PlAMV)in Imported Lily Bulbs in China
2．1 Introduction
2．2 Materials and methods
2．2．1 Field sampling
2．2．2 Materials and chemicals
2．2．3 NGS analysis and then detection by using the conventional RT-PCR assay
2．2．4 Library preparation for Strand-specific Transcriptome(rRNA-free)sequencing
2．2．5 Clustering and sequencing
2．2．6 RNAquantification and qualification
2．2．7 Total RNA extraction
2．2．8 Reverse transcription(RT)
2．2．9 RT-PCR assays for the detection of PIAMV
2．2．10 Agarose Gel Electrophoresis
2．2．11 Cloning PCR products
2．2．12 Protocol for ligation of purified PCR products
2．2．14 Rapid amplification of cDNA ends(RACE)
2．2．15 Cloning and sequencing of 5’-and 3’-ends of PlAMV from lily plants
2．2．16 Colony PCR
2．2．17 Sequencing
2．2．18 Sequence and Phylogenetie analysis using bioinformaties tools
2．3 Results
2．3．1 Viruses identified by NGS and Genome organization of PlAMV Siberia isolate
2．3．2 Phylogenetic analysis of PlAMV-Siberia isolate
2．3．3 Incidence of PlAMV in lily plants
2．4．DISCUSSION
CHAPTER THREE Development of RT-qPCR assays for the detection of Plantago asiatica mosaic virus in imported Lilium spp．in China
3．1 Introduction
3．2 Materials and methods
3．2．1 Plant materials
3．2．2 Primer and probe design for RT-qPCR assays
3．2．3 RNA extraction
3．2．5 Sensitivity and RT-qPCR assays
3．2．6 Specificity and RT-qPCR assays
3．3 Results
3．3．1 Specificity of the primer and probe
3．3．2 Standard curve and sensitivity assays
3．3．3 Detection of PlAMV in lily samples using RT-PCR and RT-qPCR assays
3．4 Discussion
CHAPTER FOUR
4．1 General Conclusions
REFERENCES
Acknowledgements
Appendix
BIOGRAPHY
