List of Contents
Abstract
摘要
List of Abbreviations
CHAPTER ONE：Literature Review
1．1 Rice importance and history
1．2 Rice viruses
1．2．1 Rice stripe virus(RSV)
1．2．2 Rice black-streaked dwarfvirus(RBSDV)
1．3 RNA silencing and plant viral diseases
1．3．1 RNA-silencing pathways in plants
1．3．2 Suppression of RNA silencing and viral counter defense
1．3．3 RNA silencing-mediated resistance to plant viruses
1．4 Agrobacterium-mediated transformation in rice
1．4．1 Advantages of Agrobacterium-mediated transformation
1．4．2 Choice of rice tissues
1．4．3 Selectable marker genes
1．4．4 Regeneration of transgenie plants
1．5 Transeriptome analysis using RNA sequencing
1．5．1 RNA-Seq for understanding plant-pathogen interactions
1．5．2 Transeriptome alteration in response to rice viruses
1．5．3 Attempts to study responses of rice to RBSDV infection
1．6 Aims and objectives of present study
References
CHAPTER TWO：Transcriptional Changes of Rice in Response to Rice Black-Streaked Dwarf Virus Infection
Abstract
2．1 Introduction
2．2 Materials and methods
2．2．1 Plant materials
2．2．2 Samples preparation
2．2．3 Mapping reads to the reference genome and annotated genes
2．2．4 Evaluation of genes from RNA-Seq and Gene Ontology(GO) analysis
2．2．5 Identification of differentially expressed genes(DEGs)and pathway analysis
2．6．Validation of RNA-Seqresults by quantitative real-time PCR(qRT-PCR)
2．3 Results
2．3．1 Confirmation of RBSDV infection in rice plants by RT-PCR
2．3．2 Transeriptome profiles
2．3．3 Identification of DEGs
2．3．4 Functional annotation of DEGs
2．3．5 KEGG enrichment analysis of DEGs
2．3．6 The RBSDV-responsive transeriptome in rice
2．3．7 Validation of RNA-Seq data by quantitative real-time PCR(qRT-PCR)
2．4 Discussion
2．5 Conclusion
References
CHAPTER THREE：Time-Course Gene Expression Analysis of Rice Plants Infected With Rice Black-Strcaked Dwarf Virus
Abstract
3．1 Introduction
3．2 Materials and methods
3．2．3 Samplings of seedlings，RNA extraction and cDNA synthesis
3．2．4 Verification of RBSDV infection in rice plants
3．2．6 Statistical analysis
3．3 Results
3．3．1 Confirmation of RBSDV infection in rice plants by RT-PCR
3．3．2 Altered expression of some genes related to dwarf symptoms
3．3．3 Different expression of some defense and stress response-related genes
3．3．4 Altered expression of some chloroplast and photosynthesis related genes
3．4 Discussion
References
CHAPTER FOUR：Overexpression of Rice Black-Streaked Dwarf Virus Non-Structural Proteins P7-1 and P9-1 Altered Expression of Some Rice Genes Targeted By Virus-Responsive MicroRNAs
Abstract
4．1 Introduction
4．2 Materials and methods
4．2．3 Transformation of rice calli
4．2．4 Detection of inserted genes via PCR
4．2．5 Verification of transgene expression by reverse-transcription PCR(RT-PCR)
4．2．6 Gene expression analysis by quantitative real-time PCR(qRT-PCR)
4．2．7 Statistical analysis
4．3 Results
4．3．2 Accumulation of overexpression constructs in transgenic plants
4．3．3 Expression of RBSDV-rexponsive miRNAs target genes in RBSDV P7-1 and P9-1-overexpressed transgenic rice
4．4 Discussion
References
CHAPTER FIVE：RNAi-mediated Resistance to Rice Black-Streaked Dwarf Virus in Transgenic Rice
Abstract
5．1．Introduction
5．2 Materials and methods
5．2．1 RBSDV RNA extraction and reverse transcription
5．2．2 Construction of plasmids
5．2．3 Transformation of rice calli
5．2．4 Detection of inserted genes via PCR
5．2．5 Selection of selectable marker free transgenic plants
5．2．6 Homozygous selection and zygosity determination
5．2．7 Assessment of transgene expression by qRT-PCR
5．2．8 Assessment of resistanee to RBSDV
5．2．9 RBSDV detection by qRT-PCR
5．3 Results
5．3．1 RNAi constructs and rice transformation
5．3．2 Accumulation of target gene-specific RNAi constructs in T0 generation
5．3．3 Segregation of the selectable marker and RNAi construct in the progeny
5．3．4 Homozygous selection and zygosity determination
5．3．5 High level resistance against RBSDV in T5 generation of transgenic rice plants that harbored either S7-2-or S8-specific RNAi
5．4 Discussion
References
Conclusions
List of Publications
Acknowledgements
声明
