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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張耀乾(Yao-Chien Alex Chang) | |
dc.contributor.author | Hsuan Pai | en |
dc.contributor.author | 白暄 | zh_TW |
dc.date.accessioned | 2021-05-16T16:26:19Z | - |
dc.date.available | 2018-03-15 | |
dc.date.available | 2021-05-16T16:26:19Z | - |
dc.date.copyright | 2013-03-15 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-02-08 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6333 | - |
dc.description.abstract | Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent viruses infecting orchids and causing economic losses worldwide. Upon virus infection, small RNA mediated antiviral RNA silencing response is activated. Such biotic stress may affect virus-specific interfering RNAs (vsRNAs) or microRNA (miRNA) regulated host gene expression. To advance our understanding of the offense-defense interactions between CymMV, ORSV and orchids, this study employed deep sequencing to analyze small RNAs from virus infected Phalaenopsis. The leaf tip-inoculation method first distinguished early and late stages of infection in non-inoculated and inoculated tissues at ten days post inoculation (dpi). Small RNA Solexa sequencing generated 11 libraries with more than five million reads each from CymMV and ORSV singly and doubly inoculated leaves and from mock-inoculated leaves. Generally, CymMV and ORSV vsRNAs were predominantly 21 and 22 nucleotides (nt), with excess positive polarity accumulating in single inoculations. While most CymMV vsRNAs were derived from RNA-dependent RNA polymerase (RdRp) coding regions, ORSV vsRNAs encompassed the coat protein coding gene and 3’-untranslated region, with a specific hotspot residing in the pseudoknot upstream to the 3’-terminal tRNA-like structure. These results suggest Dicer-like (DCL) 4 and DCL2 homologs play a leading role in mediating antivirus RNA silencing in P. amabilis using single-stranded RNA derived secondary structure as templates. Biased distribution of 5’ terminal adenosine (A), uridine (U), cytosine (C) and underrepresented 5’-guanine (G) indicate vsRNAs could be recruited into multiple Argonaute (AGO) complexes. Under mixed infection, chlorotic necrosis symptoms appeared specifically in inoculated tissues at 10 dpi, and accelerated spreading and enhanced viral titer of CymMV also occurred. The proportion of CymMV vsRNAs in total small RNAs ranged from 5.83% in singly infected tissues to 27.9% in doubly infected tissues, providing evidence of the enhancement of CymMV titer. While most vsRNA features remained unchanged in double inoculations, three additional prominent ORSV vsRNA hotspot peaks were observed. In silico prediction revealed Phalaenopsis transcript hotspots that are potential targets for vsRNA are also likely to be involved in symptom formation. The virus infection also modulated miRNA expression—for example, miR156, miR168, miR894 were up regulated and miR398, miR408, miR528 were down regulated after CymMV or ORSV infection. These infection responsive miRNAs participate in a broad spectrum of cellular processes like hormone and metabolite assimilation, signal transduction, and oxidative stress calibration. Taken together, the deep sequencing provided a global profile of vsRNAs and miRNAs in Phalaenopsis under CymMV and ORSV infection. Further research should provide valuable insights into small RNA-mediated virus-plant interactions. | en |
dc.description.provenance | Made available in DSpace on 2021-05-16T16:26:19Z (GMT). No. of bitstreams: 1 ntu-102-R99628108-1.pdf: 15031259 bytes, checksum: 01de8936123168e354521838343c5dc3 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | Contents............I
Contents of Tables............V Contents of Figures............VII Abstract............1 Introduction............3 Literature Review............6 1. The incidence of virus diseases in orchids............6 1.1. Cymbidium mosaic virus (CymMV)............6 1.2. Odontoglossum ringspot virus (ORSV)............7 1.3. Symptoms and pathogenesis of CymMV and ORSV in orchids............8 1.4. Molecular mechanisms of the synergism between CymMV and ORSV............10 2. The role of RNA silencing in plant-virus interactions............12 2.1. RNA silencing pathways in plants............12 2.2. Profiling characteristics of virus-derived small interfering RNAs (vsRNAs)............14 2.2.1. Functions of DCL proteins and size distribution of vsRNAs............15 2.2.2. Preference of 5’ terminal nucleotide of vsRNAs............16 2.2.3. Strand polarity and hotspot distribution of vsRNAs along the virus genome............17 2.3. Interactions between virus-modulated RNA silencing and the pathogenesis of viruses in plants............18 2.3.1. Roles of viral suppressors of RNA silencing (VSRs) in symptom induction............18 2.3.2. Symptom induction through vsRNA-mediated host gene regulation............19 2.3.3. Infection induced dysregulation of miRNA expression and its association in plant-virus interactions............20 Materials and Methods............22 1. Maintenance of CymMV- and ORSV-free plants............22 1.1. Plant materials and growing conditions............22 1.2. Detecting virus infection in newly purchased plants............22 1.2.1. Extracting total RNA from leaf tissues............22 1.2.2. Reverse transcription-polymerase chain reaction (RT-PCR)............23 2. Detection of CymMV and ORSV spread in P. amabilis............24 2.1. Experimental locations............24 2.2. Virus source............24 2.3. Virus inoculation and sequential sampling............25 2.4. Tissue blotting and hybridization assay for detecting virus............26 2.4.1. Tissue blotting............26 2.4.2. Preparation of digoxigenin (DIG)-labeled probes............27 2.4.3. Hybridization and chemiluminescent detection............28 3. Genome wide analysis of small RNAs from ORSV-infected P. amabilis by deep sequencing............29 3.1. ORSV inoculation assay and RNA sample preparation............29 3.2. Analyzing viral RNA and siRNA accumulation............29 3.2.1. Analyzing viral RNA accumulation by Northern blot............29 3.2.2. Analyzing viral siRNA accumulation by small RNA Northern blot............30 3.3. Small RNA deep-sequencing and annotation............31 3.3.1. cDNA library construction and sequencing............31 3.3.2. Sequencing data trimming and annotation............32 3.3.3. Target prediction for vsRNAs and miRNAs............33 3.3.3.1. Analyzing differential expressions of miRNAs............33 3.3.3.2. Prediction of putative miRNA target genes............33 3.3.3.3. Prediction of putative vsRNA target genes............34 3.4. Experimental validation of small RNAs............35 3.4.1. Validating miRNA expression by stem-loop quantitative RT-PCR (qRT-PCR)............35 3.4.2. VsRNA co-transfection assay in Oncidium protoplasts............36 3.4.2.1. Protoplast isolation............36 3.4.2.2. Co-transfection by electroporation method............37 3.4.2.3. RNA purification and Northern blot analysis............37 4. Genome wide analysis of small RNAs from CymMV and double infected P. amabilis by deep sequencing............38 4.1. CymMV and mixed inoculation assays............38 4.2. Analyzing viral RNA and siRNA accumulation............38 4.3. Construction of small RNA libraries from CymMV and double infected P. amabilis and bioinformatic analysis............39 Results............40 1. Preliminary time course assay of CymMV and ORSV infection in P. amabilis............40 1.1. ORSV infection in inoculated leaves 2 to 11 dpi............40 1.2. ORSV infection spreaded to non-inoculated tissues after 10 dpi............40 1.3. Accelerated spreading of CymMV infection in mixed inoculated leaves............41 2. Analysis of small RNAs from ORSV-infected P. amabilis by deep sequencing............42 2.1. Analysis of ORSV infection............42 2.2. Deep sequencing of small RNAs from ORSV-infected P. amabilis............43 2.3. Characteristics of ORSV viral siRNAs (vsRNAs)............45 2.3.1. ORSV vsRNA populations in inoculated (Oi) and non-inoculated (Oc) tissues............45 2.3.2. Size distribution, strand polarity, and 5’-end nucleotide preference of ORSV vsRNAs............45 2.3.3. Genome mapping, coverage and hotspots of ORSV vsRNAs............46 2.3.4. Specific vsRNA hotspot in ORSV 3’-UTR............47 2.3.5. Functional analysis of ORSV 3’-UTR hotspot vsRNA............48 2.4. Identification and analysis of conserved miRNAs............49 2.4.1. miRNA populations............49 2.4.2. Differential expressions of miRNAs in response to ORSV infection............50 2.4.3. Target prediction and possible roles of ORSV-infection responsive miRNAs............51 3. Analyses of small RNAs from CymMV and mixed infected P. amabilis by deep sequencing............53 3.1. Analysis of CymMV and mixed infection............53 3.2. Deep sequencing of small RNAs from CymMV and mixed infected P. amabilis............55 3.3. Characteristics of vsRNAs in CymMV and doubly infected tissues............56 3.3.1 VsRNA populations in singly and doubly infected tissues............56 3.3.2. Characteristics of vsRNAs in CymMV and double infected tissues............57 3.3.3. Genome mapping and coverage of CymMV vsRNAs............58 3.3.4. Specific ORSV vsRNA hotspots occurred in mixed infected tissues............59 3.3.5. Prediction of potential P. amabilis target transcripts of vsRNAs............59 3.4. Identification and analysis of conserved miRNAs in CymMV and mixed infected Phalaenopsis amabilis............61 3.4.1. miRNA populations............61 3.4.2. Differential expressions and predicted targets of CymMV and double infection responsive miRNAs............62 Discussion............64 1. Synergistic enhancement of CymMV infection by ORSV co-inoculation in Phalaenopsis amabilis............64 2. The leading roles of DCL4 and DCL2 in P. amabilis............66 3. Asymmetrical strand polarity and 5’-end nucleotide identity of CymMV and ORSV vsRNAs............66 4. Differential distribution of CymMV and ORSV vsRNA along viral genomes............68 5. VsRNA-mediated host gene silencing may underly the mechanism of symptom formation............70 6. Roles of infection responsive miRNAs in Phalaenopsis-virus interactions............71 7. The involvement of novel miRNAs and other small RNAs in response to viral stresses............74 Conclusion and Future Directions............76 References............78 | |
dc.language.iso | en | |
dc.title | 以高通量定序方式分析蝴蝶蘭感染齒舌蘭輪斑病毒及蕙蘭嵌紋病毒後小型核醣核酸組成之變化 | zh_TW |
dc.title | Genome Wide Analysis of Small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus Infected Phalaenopsis by Deep Sequencing | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-1 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 林納生(Na-Sheng Lin) | |
dc.contributor.oralexamcommittee | 徐堯煇(Yau-Heiu Hsu),胡仲祺(Chung-Chi Hu),張清安(Ching-An Chang) | |
dc.subject.keyword | 菸草鑲嵌病毒屬,馬鈴薯X病毒屬,微型核醣核酸,病毒小型干擾核醣核酸, | zh_TW |
dc.subject.keyword | Tobamovirus,Potexvirus,microRNA,viral small-interfering RNA, | en |
dc.relation.page | 191 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2013-02-08 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 園藝學研究所 | zh_TW |
顯示於系所單位: | 園藝暨景觀學系 |
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