介体灰飞虱蛋白与水稻条纹病毒-NCP互作验证及VAP-B在病毒传播功能的初步分析
发布时间：2024-12-17 22:02
水稻生长发育过程中会受到多种病害的影响,其中条纹叶枯病在水稻产区多次间歇性暴发,对水稻的生产构成严重威胁。该病害常发生于温带和亚热带地区,21世纪初在中国稻麦轮作区引起高达30%-40%的减产。水稻条纹叶枯病的病原是水稻条纹病毒(RSV),该病毒依靠介体灰飞虱(SBPH,Laodelphax striatellus)以持久增殖的方式水平传播,并且还能经过灰飞虱的卵进行垂直传播。灰飞虱不仅可以取食水稻、小麦、燕麦、玉米和大麦等多种禾本科作物,同时也是RSV等重要植物病毒的传播介体。RSV-SBPH的相互作用是一个复杂的机制,决定着病毒能否在自然界中成功传播,并进一步影响病害的暴发流行。病毒在介体内的侵染、运动和复制涉及到了大量的蛋白质间相互作用。本研究利用酵母双杂交技术明确了3种介体灰飞虱蛋白与RSV NCP之间的相互作用,这3种蛋白分别为灰飞虱糖转运蛋白1、灰飞虱糖转运蛋白2和囊泡相关膜蛋白相关蛋白B(VAP-B),并对这3种蛋白在介体灰飞虱内的mRNA表达水平进行了检测。同时进一步利用pull-down实验证实了VAP-B与RSV NCP之间存在互作。随后利用昆虫的RNAi技术研究了V...
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
中文摘要
Abstract
CHAPTER-Ⅰ Literature review
1.1 Economic importance of rice
1.2 Rice stripe disease and its damage for rice
1.2.1 Symptomology and alternate host plants
1.3 Rice stripe virus
1.4 Significance of plant viruses transmitted by planthoppers
1.5 Laodelphax striatellus(SBPH):The vector of RSV
1.5.1 Biological characteristics
1.5.2 Distribution
1.5.3 Occurrence in China
1.6 Virus transmission mechanisms by insect vectors
1.6.1 Persistent propagative virus transmission
1.6.2 The transmission mechanism of tenuiviruses
1.6.3 Transmission route of rice stripe virus
1.7 A general overview of protein-protein interactions
1.7.1 Yeast two-hybrid system
1.7.2 A brief introduction to other protein-protein interaction verification methods
1.8 Purpose and significance of the research
CHAPTER-Ⅱ ST1,ST2 and VAP-B amplification and analysis
2.1 Materials and Methods
2.1.1 Total RNA Extraction
2.1.2 Reverse transcription and amplification of ST1,ST2 and VAP-B genes
2.1.3 Purification of PCR products
2.1.4 Ligation of purified DNA into the p MD-19T vector
2.1.5 Transformation of ligated product into E.coli
2.1.6 Plasmid extraction
2.1.7 Biological interpretation of ST1,ST2 and VAP-B genes
2.2 Results and analysis
2.2.1 Total RNA Extraction from SBPH
2.2.2 Amplification and Blast analysis of ST1,ST2 and VAP-B genes
2.2.3 Domain analysis and multiple sequence alignment of ST1,ST2 and VAP-B genes
2.2.4 Transmembrane prediction and phylogenetic analysis of ST1,ST2 and VAP-B genes
2.3 Discussion
CHAPTER-Ⅲ Verification of interaction between Ls ST1,Ls ST2 and VAP-B with RSV-NCP by Yeast two-hybrid
3.1 Materials and Methods
3.1.1 Yeast two-hybrid system to verify the interaction between RSV-NCP and Ls ST1,Ls ST2,and VAP-B
3.2 Results
3.2.1 Vector construction
3.2.2 Functional assay of Ls ST1,Ls ST2 and VAP-B(prey)with RSV-NCP(bait)in co-transfected yeast cells
3.3 Discussion
CHAPTER-Ⅳ mRNA expression level analysis by reverse transcription quantitative real-time PCR
4.1 Materials and Methods
4.1.1 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.2 Results
4.2.1 Primers constructed for RT-q PCR analysis
4.2.2 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.3 Discussion
5 CHAPTER-Ⅴ Validation of interaction between VAP-B with RSV-NCP by Pull-Down Assay
5.1 Materials and Methods
5.1.1 Pull-down assay to verify the interaction between VAP-B and RSV-NCP
5.2 Results
5.2.1 Vector construction
5.2.2 Pull-down assay
5.3 Discussion
CHAPTER-Ⅵ Effect of RNA interference of VAP-B on infection and relative expression of RSV in SBPH
6.1 Materials and Methods
6.1.1 ds RNA synthesis
6.1.2 Microinjection of the ds RNA to the SBPH
6.1.3 Effect of RNAi of VAP-B on infection and relative expression of RSV
6.1.4 Effect of RNAi of VAP-B on the transmission of RSV
6.2 Results
6.2.1 ds RNA construction
6.2.2 Effect of ds VAP-B injection on the expression of VAP-B in the SBPH
6.2.3 Effect of RNAi of VAP-B on infection and relative expression of RSV in the viruliferous SBPH
6.2.4 Effect of RNAi of VAP-B on the transmission of RSV
6.3 Discussion
CHAPTER-Ⅶ First reports of Tomato chlorosis virus and Potato leafroll virus in tomato from Pakistan
7.1 Materials and Methods
7.1.1 Samples collection
7.1.2 ELISA
7.1.3 RNA isolation and RT-PCR
7.1.4 Whitefly transmission
7.1.5 Next-Generation Sequencing
7.1.6 BLAST and phylogenetic analysis
7.1.7 Selection pressure and recombination analysis
7.2 Results
7.2.1 ELISA for different viruses
7.2.2 RT-PCR
7.2.3 Transmission test
7.2.4 Sequencing,BLAST and Phylogenetic analysis
7.2.5 Full genome sequence analysis
7.2.6 Selection pressure and recombination analysis
7.2.7 Gene flow and genetic differentiation analysis
7.2.8 Haplotype and nucleotide diversity
7.3 Discussion
CHAPTER-Ⅷ Summary
Bibliography
Acknowledgement
Author's Biography
Appendix Ⅰ
Appendix Ⅱ
本文编号：4016595
【文章页数】：131 页
【学位级别】：博士
【文章目录】：
中文摘要
Abstract
CHAPTER-Ⅰ Literature review
1.1 Economic importance of rice
1.2 Rice stripe disease and its damage for rice
1.2.1 Symptomology and alternate host plants
1.3 Rice stripe virus
1.4 Significance of plant viruses transmitted by planthoppers
1.5 Laodelphax striatellus(SBPH):The vector of RSV
1.5.1 Biological characteristics
1.5.2 Distribution
1.5.3 Occurrence in China
1.6 Virus transmission mechanisms by insect vectors
1.6.1 Persistent propagative virus transmission
1.6.2 The transmission mechanism of tenuiviruses
1.6.3 Transmission route of rice stripe virus
1.7 A general overview of protein-protein interactions
1.7.1 Yeast two-hybrid system
1.7.2 A brief introduction to other protein-protein interaction verification methods
1.8 Purpose and significance of the research
CHAPTER-Ⅱ ST1,ST2 and VAP-B amplification and analysis
2.1 Materials and Methods
2.1.1 Total RNA Extraction
2.1.2 Reverse transcription and amplification of ST1,ST2 and VAP-B genes
2.1.3 Purification of PCR products
2.1.4 Ligation of purified DNA into the p MD-19T vector
2.1.5 Transformation of ligated product into E.coli
2.1.6 Plasmid extraction
2.1.7 Biological interpretation of ST1,ST2 and VAP-B genes
2.2 Results and analysis
2.2.1 Total RNA Extraction from SBPH
2.2.2 Amplification and Blast analysis of ST1,ST2 and VAP-B genes
2.2.3 Domain analysis and multiple sequence alignment of ST1,ST2 and VAP-B genes
2.2.4 Transmembrane prediction and phylogenetic analysis of ST1,ST2 and VAP-B genes
2.3 Discussion
CHAPTER-Ⅲ Verification of interaction between Ls ST1,Ls ST2 and VAP-B with RSV-NCP by Yeast two-hybrid
3.1 Materials and Methods
3.1.1 Yeast two-hybrid system to verify the interaction between RSV-NCP and Ls ST1,Ls ST2,and VAP-B
3.2 Results
3.2.1 Vector construction
3.2.2 Functional assay of Ls ST1,Ls ST2 and VAP-B(prey)with RSV-NCP(bait)in co-transfected yeast cells
3.3 Discussion
CHAPTER-Ⅳ mRNA expression level analysis by reverse transcription quantitative real-time PCR
4.1 Materials and Methods
4.1.1 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.2 Results
4.2.1 Primers constructed for RT-q PCR analysis
4.2.2 mRNA expression level analysis in different tissues of SBPH by RT-q PCR
4.3 Discussion
5 CHAPTER-Ⅴ Validation of interaction between VAP-B with RSV-NCP by Pull-Down Assay
5.1 Materials and Methods
5.1.1 Pull-down assay to verify the interaction between VAP-B and RSV-NCP
5.2 Results
5.2.1 Vector construction
5.2.2 Pull-down assay
5.3 Discussion
CHAPTER-Ⅵ Effect of RNA interference of VAP-B on infection and relative expression of RSV in SBPH
6.1 Materials and Methods
6.1.1 ds RNA synthesis
6.1.2 Microinjection of the ds RNA to the SBPH
6.1.3 Effect of RNAi of VAP-B on infection and relative expression of RSV
6.1.4 Effect of RNAi of VAP-B on the transmission of RSV
6.2 Results
6.2.1 ds RNA construction
6.2.2 Effect of ds VAP-B injection on the expression of VAP-B in the SBPH
6.2.3 Effect of RNAi of VAP-B on infection and relative expression of RSV in the viruliferous SBPH
6.2.4 Effect of RNAi of VAP-B on the transmission of RSV
6.3 Discussion
CHAPTER-Ⅶ First reports of Tomato chlorosis virus and Potato leafroll virus in tomato from Pakistan
7.1 Materials and Methods
7.1.1 Samples collection
7.1.2 ELISA
7.1.3 RNA isolation and RT-PCR
7.1.4 Whitefly transmission
7.1.5 Next-Generation Sequencing
7.1.6 BLAST and phylogenetic analysis
7.1.7 Selection pressure and recombination analysis
7.2 Results
7.2.1 ELISA for different viruses
7.2.2 RT-PCR
7.2.3 Transmission test
7.2.4 Sequencing,BLAST and Phylogenetic analysis
7.2.5 Full genome sequence analysis
7.2.6 Selection pressure and recombination analysis
7.2.7 Gene flow and genetic differentiation analysis
7.2.8 Haplotype and nucleotide diversity
7.3 Discussion
CHAPTER-Ⅷ Summary
Bibliography
Acknowledgement
Author's Biography
Appendix Ⅰ
Appendix Ⅱ
本文编号：4016595
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/4016595.html
最近更新
教材专著
