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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張雅君 | |
| dc.contributor.author | Shu-Chuan Lee | en |
| dc.contributor.author | 李淑娟 | zh_TW |
| dc.date.accessioned | 2021-06-13T15:58:27Z | - |
| dc.date.available | 2013-06-05 | |
| dc.date.copyright | 2008-06-05 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-05-27 | |
| dc.identifier.citation | Ashby, J., Boutant, E., Seemanpillai, M., Sambade, A., Ritzenthaler, C., & Heinlein, M. (2006). Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein. Journal of Virology, 80, 8329-8344.
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Deom, C. M., & He, X. Z. (1997). Second-site reversion of a dysfunctional mutation in a conserved region of the tobacco mosaic tobamovirus movement protein. Virology, 232, 13-18. Ding, X. S., Liu, J., Cheng, N. H., Folimonov, A., Hou, Y. M., Bao, Y., Katagi, C., Carter, S. A., & Nelson, R. S. (2004). The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing. Molecular Plant Microbe Interaction, 17, 583-592. Fenczik, C. A., Padgett, H. S., Holt, C. A., Casper, S. J., & Beachy, R. N. (1995). Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant. Molecular Plant Microbe Interaction, 8, 666-673. Hilf, M. E., & Dawson, W. O. (1993). The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement. Virology, 193, 106-114. Hirashima, K., & Watanabe, Y. (2001). Tobamovirus replicase coding region is involved in cell-to-cell movement. Journal of Virology, 75, 8831-8836. Hirashima, K., & Watanabe, Y. (2003). RNA helicase domain of tobamovirus replicase executes cell-to-cell movement possibly through collaboration with its nonconserved region. Journal of Virology, 77, 12357-12362. Hofmann, C., Sambade, A., & Heinlein, M. (2007). Plasmodesmata and intercellular transport of viral RNA. Biochemical Society Transactions, 35, 142-145. Ikegami, M., Isomura, Y., Matumoto, Y., Chatani, M., & Inouye, N. (1995). The complete nucleotide sequence of odontoglossum ringspot virus (Cy-1 strain) genomic RNA. Microbiology and Immunology, 39, 995-1001. Kragler, F., Curin, M., Trutnyeva, K., Gansch, A., & Waigmann, E. (2003). MPB2C, a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein. Plant Physiology, 132, 1870-1883. Kubota, K., Tsuda, S., Tamai, A., & Meshi, T. (2003). Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. Journal of Virology, 77, 11016-11026. Lucas, W. J. (2006). Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes. Virology, 344, 169-184. Meshi, T., Watanabe, Y., Saito, T., Sugimoto, A., Maeda, T., & Okada, Y. (1987). Function of the 30 kd protein of tobacco mosaic virus: involvement in cell-to-cell movement and dispensability for replication. Embo Journal, 6, 2557-2563. Rabindran, S., Robertson, C., Achor, D., German-Retana, S., Holt, C. A., & Dawson, W. O. (2005). Odontoglossum ringspot virus host range restriction in Nicotiana sylvestris maps to the replicase gene. Molecular Plant Pathology, 6, 439-447. Ryu, K. H., & Park, W. M. (1995). The complete nucleotide sequence and genome organization of odontoglossum ringspot tobamovirus RNA. Archives of Virology, 140, 1577-1587. Saito, T., Yamanaka, K., & Okada, Y. (1990). Long-distance movement and viral assembly of tobacco mosaic virus mutants. Virology, 176, 329-336. Waigmann, E., Lucas, W. J., Citovsky, V., & Zambryski, P. (1994). Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability. Proceedings of National Academica Science U S A, 91, 1433-1437. Waigmann, E., Chen, M. H., Bachmaier, R., Ghoshroy, S., & Citovsky, V. (2000). Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. EMBO Journal, 19, 4875-4884. Wang, H. H., Yu, H. H., & Wong, S. M. (2004). Mutation of Phe50 to Ser50 in the 126/183-kDa proteins of Odontoglossum ringspot virus abolishes virus replication but can be complemented and restored by exact reversion. Journal of General Virology, 85, 2447-2457. Yu, H. H., & Wong, S. M. (1998). A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene. Archives of Virology, 143, 163-171. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38044 | - |
| dc.description.abstract | 蘭花為台灣重要的經濟花卉，而病毒感染為蘭花產業上的一大威脅。為早期篩檢罹病毒植株，本論文針對感染蘭科植物的重要病毒：蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)開發了三種不同的檢測法，以供有不同的需求情況時可以選擇使用。第一個方法為利用多對引子反轉錄聚合酶連鎖反應(multiplex RT-PCR)進行病毒檢測。分別針對CymMV和ORSV設計專一性引子對，以增幅個別病毒的鞘蛋白基因。除此之外，也針對植物粒線體煙醯胺腺嘌呤二核酸去氫酶(NADH dehydrogenase, nad5)基因設計另一組引子對，以此作為檢測時的RT-PCR反應內在對照組。利用nad5專一性引子對，不論在健康或是病毒感染的植物全RNA樣品中，皆可以穩定的增幅出nad5 mRNA的cDNA片段。上述三組引子對不論是利用單對引子及多對引子反轉錄聚合酶連鎖反應進行測試，結果證實皆具有高度專一性。當以多對引子反轉錄聚合酶連鎖反應測試方法的靈敏度時，不論樣品含有單一或兩種病毒，針對CymMV檢測靈敏度皆可達1 pg，對ORSV的檢測靈敏度則為10 pg。在複合感染的樣品中，此二病毒存在量的差異似乎不會影響檢測的結果。第二個方法為利用細菌系統產生病毒鞘蛋白，用以生產專一性的抗體，利用此抗體進行I-ELISA檢測。在本實驗中產生的抗體稱為HM-Cy及HM-OR，當利用西方轉漬法(western blot)進行分析時，分別對CymMV及ORSV鞘蛋白有高專一性反應，同時對於健康的植物組織則不反應，具有相當低的背景值。這樣的特性有利於在I-ELISA檢測中，樣品呈現正反應或負反應的判讀。第三個方法則是綜合了第一和第二方法的優點，稱為免疫捕捉-多對引子反轉錄聚合酶連鎖反應(multiplex immunocapture RT-PCR)。將純化的兩種IgG抗體附著在聚苯乙烯PCR反應管中，利用此附著的抗體吸附散佈在植物汁液中的CymMV與ORSV病毒顆粒，再經由加熱步驟釋放病毒RNA到反應溶液中，針對個別病毒的鞘蛋白附近序列，選擇高度保守的區域來設計引子對， 再經由多對引子反轉錄聚合酶連鎖反應進行病毒基因體片段的增幅。結果顯示，經由此方法可以增加檢測的靈敏度，相較於I-ELISA約增加25至125倍的靈敏度，而且可以在同一反應管中同時進行兩種病毒的檢測。這些檢測方法的研發可以應用於例行的病毒篩檢，期望對於國內蘭花種苗病毒驗證作業有所助益。除此之外，為了進一步了解病毒本身的特性，本研究也進行了ORSV全長度感染性病毒株的構築，所篩選出的感染性病毒株，分析其序列全長為6611核苷酸，具有四個開放解讀框。將此全長序列與其他已發表的ORSV全長序列進行序列比對，其序列的相同度在核苷酸部分高達82-100%，而胺基酸序列則有94-100%的相同度。在分析不同的ORSV分離株時發現，在其移動蛋白5’端皆具有兩個轉譯起始碼，然而不同系統對於此轉譯起始碼的取決不同，因此不知移動蛋白真正的起始位置為何，然而在本實驗中所構築的全長度感染性病毒株只具有一個轉譯起始碼，只能產生分子量較小的移動蛋白(約31 kDa)，在此全長度感染性病毒株中，其感染植物的表現、病毒量累積及病徵型態皆與野生型ORSV相同，意即此31 kDa的移動蛋白即足以負責ORSV相對應的所有功能。再者，由篩選全長度感染性病毒株中發現，有些具有複製能力的選殖株不具有系統性感染菸草植株的能力，藉由分析其全長序列，發現有數個突變存在其中，這些突變所造成的胺基酸改變分別位於複製酵素、移動蛋白及鞘蛋白。排列組合這些突變的胺基酸，再進行接種分析。結果顯示，這些突變的排列組合不會影響全長度感染性病毒株在菸草原生質體中的複製能力。然而，全長度感染性病毒株在植物中細胞間的移動會受到移動蛋白中Met97突變成Val97的影響，以至於在移動行為上有缺陷。而在移動蛋白上，另外一個位置由Thr49突變成Ile49則會部分恢復移動蛋白的功能。鞘蛋白上的突變則對病毒的細胞間移動能力沒有影響。但是，鞘蛋白再病毒的系統性長距離移動具有決定性的影響力，如果鞘蛋白的Glu100突變成Gly100，則此全長度感染性病毒株會失去系統性長距離移動的能力。 | zh_TW |
| dc.description.abstract | Orchid is one of the most important commercial plants in Taiwan. Virus infection in the mass production of orchid may result in great economic loss. Detection and screening the virus infected plant will reduce the risk of mass production and increase the plant quality. Three detection methods were developed for the detection of two covalent and important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). The first one was a multiplex RT-PCR method. Specific primers were designed based on the respective viral coat protein genes. In addition, one primer pair derived from the plant mitochondrial NADH dehydrogenase gene (nad5) was used as an internal control amplified in the multiplex RT-PCR. Application of this multiplex RT-PCR could greatly reduce the cost and false negative results in the routine detection. The second method was I-ELISA detection using antisera against by purified viral capsid proteins expressed in bacteria. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were validated by immunoblotting and both of them showed low background reactivity to healthy samples, especially when compared with the commercially available anti-ORSV antibody. Furthermore, they had a higher S/H ratio (sample OD405/healthy control OD405) than commercial antibodies in tested orchids. The third method was so-called a multiplex immunocapture-reverse transcription-polymerase chain reaction (mIC-RT-PCR) which combines the advantages of ELISA and mRT-PCR. Purified HM-Cy IgG and HM-OR IgG were coated onto the polystyrene tubes to enrich viral particles from plant extracts. After heating, viral RNAs were released and followed by the multiplex RT-PCR (mRT-PCR) amplification and gel electrophoresis. The simultaneous detection of CymMV and ORSV was successfully performed by mIC-RT-PCR in single- or mixed-infection samples. The sensitivity of mIC-RT-PCR was 25 - 125 times higher than I-ELISA. These three detection methods provide more options according to different requests. To investigate the viral biological properties, the ORSV full-length cDNA clones were constructed under the driven of a T7 promoter. A full-length cDNA clone, pORSV-7, with 6611 nt containing four open reading frames showing a systemically infection, was analyzed and compared with six reported ORSV isolates. The sequence homology of different ORSV isolates was ranged from 82 to 99% in nucleotides and 94 to 100% in amino acids. The ORSV MP was somehow confusing due to its different annotation of MP ORF; however, our data suggested that the product of ORSV-7 MP ORF which only containing 840 nucleotides (279 amino acids) is sufficient for virus function. Comparisons of two full-length cDNA clones, pORSV-2 and -7, we found differences in five amino acids including one in replicase protein, two in movement protein, and two in capsid proteins, respectively. Chimeric cDNA clones were generated to investigate the viral movement determinants. All chimeric constructs could replicate in Nicotiana benthamiana protoplasts at the similar level. Inoculation tests with different combinations of MP and CP of pORSV-2 and -7 revealed a complementary interaction in ORSV long-distance movement. We further narrow down the determinants of cell-to-cell movement in Chenopodium quinoa plants to the Met97 in the MP of ORSV-7. The mutation of Thr49 to Ile49 in the MP of ORSV-2 complement the Met97 to Val97 mutant of ORSV-7 to rescue its movement. CP was dispensable for ORSV cell-to-cell movement, but is important for long-distance movement. The sufficient long-distance movement of ORSV in N. benthamiana was mapped to the Glu100 in CP of ORSV-7. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T15:58:27Z (GMT). No. of bitstreams: 1 ntu-97-D91633003-1.pdf: 3104988 bytes, checksum: 552ab12e94964f2364ab748f6fef4cb4 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 中文摘要 ii
Abstract iv Introduction 1 Characterization and production of orchids 1 Cymbidium mosaic virus (CymMV) 2 Odontoglossum ringspot virus (ORSV) 2 Detection of CymMV and ORSV 3 Biological properties of ORSV 4 Objectives of this study 5 Chapter I 7 Multiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA 7 Abstract 9 Introduction 10 Materials and methods 13 Virus and plant materials 13 Plant total RNA and viral RNA extraction 13 Primer design 14 Simplex reverse transcription-polymerase chain reaction (RT-PCR) 15 Multiplex RT-PCR 16 Agarose gel electrophoresis 16 Results 18 Specificity of designed primers 18 Optimization of multiplex RT-PCR reaction 19 Detection sensitivity of multiplex RT-PCR 20 Detection of orchid samples from market using multiplex RT-PCR 21 Discussions 23 Literature cited 27 Table 33 Table 1. Primer names, sequences and position in the respective genomes, and expected size of RT-PCR product for each primer pair 33 Figures 34 Fig. 1. The specificity of each primer pair in simplex and multiplex RT-PCR. 34 Fig. 2. Optimization of multiplex RT-PCR reaction with different concentration ratios of primer set. 35 Fig. 3. Detection sensitivity of multiplex RT-PCR. 36 Fig. 4. Multiplex RT-PCR detection with different amounts of two viral RNAs. 37 Fig. 5. Simultaneous detection of CymMV and ORSV in orchid plants using multiplex RT-PCR. 38 Chapter II 39 Performances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus 39 Abstract 41 Introduction 42 Materials and methods 45 Virus source and field sample collection 45 Extraction of plant total RNA 45 Reverse transcription-polymerase chain reaction (RT-PCR) 46 Construction and expression of the recombinant CymMV and ORSV CP genes 47 Indirect-ELISA (I-ELISA) 49 Immunoblot analysis 50 Results 52 Expression of recombinant CymMV and ORSV CPs for antiserum production 52 Specificity of home-made CymMV and ORSV antisera (HM-Cy and HM-OR) analyzed by immunoblot 53 Detection sensitivity of HM-Cy and HM-OR compared with commercial antibodies 53 Field survey for the incidence of CymMV and ORSV in Taiwan 55 Discussion 57 References 61 Tables 61 Table 1 Indirect-ELISA test of the home-made CymMV and ORSV antisera (HM-Cy and HM-OR) produced by E. coli-expressed recombinant capsid proteins and antibodies purchased from Agdia Inc. (A-Cy and A-OR) 67 Table 2 Number of samples (% in parenthesis) of orchid plants collected from different farms testing positive for CymMVand ORSV by I-ELISA using HM-Cy and HM-OR antisera simultaneously 68 Figures 69 Fig. 1 Amplification of CymMV and ORSV CP genes from total RNA of diseased orchid by RT-PCR. 69 Fig. 2 Bacterial lysates of E. coli BL21 (DE3) transformed with pET29a(+)-CyCP (a) and pET29a(+)-ORCP (b) were analyzed in a 12% SDS-polyacrylamide gel. 70 Fig. 3 Specificity of home-made CymMV and ORSV antisera (HM-Cy and HM-OR) analyzed by immunoblot. 71 Fig. 4 Detection sensitivities of home-made and commercial antibodies against CymMV and ORSV. 73 Chapter III 75 Development of multiplex immunocapture-RT-PCR for simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids 75 Abstract 77 Introduction 79 Materials and methods 82 Virus source 82 Primer design 82 Extraction of plant total RNA 83 Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) 83 Multiplex immunocapture-RT-PCR (mIC-RT-PCR) 83 Indirect-ELISA (I-ELISA) 84 Results 85 Specificity of designed primer sets 85 Detection of CymMV and ORSV by mIC-RT-PCR 85 Comparison of I-ELISA and mIC-RT-PCR 86 Evaluation of mIC-RT-PCR 87 Discussion 88 References 90 Tables 94 Table 1. Primer names, sequences and position in the respective genomes, and expected size of RT-PCR product for each primer pair 94 Figures 95 Fig. 1 Specificity of primer SetI (CymMV-UF+CymMV-UR) and SetII (ORSV-UF+ORSV-UR) tested by multiplex RT-PCR. 95 Fig. 3 Sensitivity test of I-ELISA using polyclonal antisera: HM-Cy and HM-OR. 97 Fig. 5 Evaluation assay of mIC-RT-PCR detection method. 99 Chapter IV 101 Construction and characterization of Odontoglussum ringspot virus infectious cDNA clone 101 Abstract 103 Introduction 104 Materials and methods 107 Virus source and plants 107 Extraction of plant total RNA 107 Reverse transcription-polymerase chain reaction (RT-PCR) 108 Plasmid construction 109 in vitro transcription 109 Inoculation of N. benthamiana protoplasts and plants 110 Preparation of DIG-labeled probe 110 Northern blot analysis 111 Immunoblot analysis 111 Sequence comparison 112 Results 113 Construction of T7 promoter-derived ORSV full-length cDNA clone 113 Infectivity assay of ORSV full-length cDNA clones 113 Complete sequence of ORSV infectious cDNA clone, pORSV-7 114 Sequence comparison of ORSV-7 and other ORSV isolates 115 The translation start site of ORSV MP ORF 116 Discussions 118 References 121 Tables 126 Table 1. Primers used in this study 126 Table 2. Percentage of sequence identity (%) between ORSV-7 and other ORSV isolates 127 Figures 128 Fig. 1 Schematic representation of genome organization of ORSV. 128 Fig. 2 The infectivity assay of ORSV full-length cDNA clones in Nicotiana benthamiana protoplasts. 129 Fig. 3 The symptom produced by ORSV infectious cDNA clones in N. benthamiana plants. 130 Fig. 5 Complete sequence of infectious cDNA clone, pORSV-7, and its encoded amino acid sequences for each ORF. 134 Fig. 6 The sequence comparison of predicted MP translation start sites in different ORSV isolates. 135 Chapter V 137 The movement determinants of Odontoglossum ringspot virus 137 Abstract 139 Introduction 140 Materials and methods 143 Construction of chimeric mutants 143 Protoplast preparation and inoculation, Northern and western blot analysis of inoculated plants 143 Results 144 Differential movement abilities of infectious cDNA clones pORSV-2 and -7 144 The nucleotide and amino acid changes between pORSV-7 and -2 145 The determinants of ORSV cell-to-cell movement 146 The determinants of ORSV long-distance movement 148 Discussions 152 References 155 Figures 160 Fig.1 Schematic representation of the ORSV genome of infectious cDNA clones, pORSV-2, pORSV-7, and derivative constructions. 161 Fig. 2 The infectivity of ORSV infectious cDNA clones in N. benthamiana. 162 Fig. 3 Symptoms induced by ORSV wild type and infectious cDNA clones in local lesion and systemic hosts. 163 Fig. 4 The infectivity of ORSV infectious cDNA clones and their derivatives in N. benthamiana protoplasts. 164 Fig. 5 Symptoms induced by ORSV infectious cDNA clones and their derivatives in local lesion C. quinoa. 165 Fig. 6 The CP of ORSV was dispensable for it cell-to-cell movement. 166 Fig. 7 The systemic movement of ORSV infectious cDNA clones and their derivatives in N. benthamiana plants. 168 | |
| dc.language.iso | en | |
| dc.subject | full-length infectious cDNA clone | en |
| dc.subject | orchid | en |
| dc.subject | CymMV | en |
| dc.subject | ORSV | en |
| dc.subject | virus detection | en |
| dc.title | 兩種感染蘭科植物重要病毒之檢測法開發及齒舌蘭輪斑病毒感染性選殖株之構築與特性分析 | zh_TW |
| dc.title | Development of detection methods for two important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), and construction and characterization of an ORSV infectious cDNA clone | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 蔡慶修,王昭雯,劉瑞芬,沈湯龍 | |
| dc.subject.keyword | 蘭花,蕙蘭嵌紋病毒,齒舌蘭輪斑病毒,病毒檢測,全長度感染性病毒株, | zh_TW |
| dc.subject.keyword | orchid,CymMV,ORSV,virus detection,full-length infectious cDNA clone, | en |
| dc.relation.page | 168 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-05-28 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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