張雅君臺灣大學:植物病理與微生物學研究所李淑娟Lee, Shu-ChuanShu-ChuanLee2010-05-112018-06-292010-05-112018-06-292008U0001-2605200817510100http://ntur.lib.ntu.edu.tw//handle/246246/181924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。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.中文摘要 iibstract ivntroduction 1haracterization and production of orchids 1ymbidium mosaic virus (CymMV) 2dontoglossum ringspot virus (ORSV) 2etection of CymMV and ORSV 3iological properties of ORSV 4bjectives of this study 5hapter I 7ultiplex RT-PCR detection of two orchid viruses with an internal control of plant nad5 mRNA 7bstract 9ntroduction 10aterials and methods 13irus and plant materials 13lant total RNA and viral RNA extraction 13rimer design 14implex reverse transcription-polymerase chain reaction (RT-PCR) 15ultiplex RT-PCR 16garose gel electrophoresis 16esults 18pecificity of designed primers 18ptimization of multiplex RT-PCR reaction 19etection sensitivity of multiplex RT-PCR 20etection of orchid samples from market using multiplex RT-PCR 21iscussions 23iterature cited 27able 33able 1. Primer names, sequences and position in the respective genomes, and expected size of RT-PCR product for each primer pair 33igures 34ig. 1. The specificity of each primer pair in simplex and multiplex RT-PCR. 34ig. 2. Optimization of multiplex RT-PCR reaction with different concentration ratios of primer set. 35ig. 3. Detection sensitivity of multiplex RT-PCR. 36ig. 4. Multiplex RT-PCR detection with different amounts of two viral RNAs. 37ig. 5. Simultaneous detection of CymMV and ORSV in orchid plants using multiplex RT-PCR. 38hapter II 39erformances and application of antisera produced by recombinant capsid proteins of Cymbidium mosaic virus and Odontoglossum ringspot virus 39bstract 41ntroduction 42aterials and methods 45irus source and field sample collection 45xtraction of plant total RNA 45everse transcription-polymerase chain reaction (RT-PCR) 46onstruction and expression of the recombinant CymMV and ORSV CP genes 47ndirect-ELISA (I-ELISA) 49mmunoblot analysis 50esults 52xpression of recombinant CymMV and ORSV CPs for antiserum production 52pecificity of home-made CymMV and ORSV antisera (HM-Cy and HM-OR) analyzed by immunoblot 53etection sensitivity of HM-Cy and HM-OR compared with commercial antibodies 53ield survey for the incidence of CymMV and ORSV in Taiwan 55iscussion 57eferences 61ables 61able 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) 67able 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 68igures 69ig. 1 Amplification of CymMV and ORSV CP genes from total RNA of diseased orchid by RT-PCR. 69ig. 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. 70ig. 3 Specificity of home-made CymMV and ORSV antisera (HM-Cy and HM-OR) analyzed by immunoblot. 71ig. 4 Detection sensitivities of home-made and commercial antibodies against CymMV and ORSV. 73hapter III 75evelopment of multiplex immunocapture-RT-PCR for simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids 75bstract 77ntroduction 79aterials and methods 82irus source 82rimer design 82xtraction of plant total RNA 83ultiplex reverse transcription-polymerase chain reaction (mRT-PCR) 83ultiplex immunocapture-RT-PCR (mIC-RT-PCR) 83ndirect-ELISA (I-ELISA) 84esults 85pecificity of designed primer sets 85etection of CymMV and ORSV by mIC-RT-PCR 85omparison of I-ELISA and mIC-RT-PCR 86valuation of mIC-RT-PCR 87iscussion 88eferences 90ables 94able 1. Primer names, sequences and position in the respective genomes, and expected size of RT-PCR product for each primer pair 94igures 95ig. 1 Specificity of primer SetI (CymMV-UF+CymMV-UR) and SetII (ORSV-UF+ORSV-UR) tested by multiplex RT-PCR. 95ig. 3 Sensitivity test of I-ELISA using polyclonal antisera: HM-Cy and HM-OR. 97ig. 5 Evaluation assay of mIC-RT-PCR detection method. 99hapter IV 101onstruction and characterization of Odontoglussum ringspot virus infectious cDNA clone 101bstract 103ntroduction 104aterials and methods 107irus source and plants 107xtraction of plant total RNA 107everse transcription-polymerase chain reaction (RT-PCR) 108lasmid construction 109n vitro transcription 109noculation of N. benthamiana protoplasts and plants 110reparation of DIG-labeled probe 110orthern blot analysis 111mmunoblot analysis 111equence comparison 112esults 113onstruction of T7 promoter-derived ORSV full-length cDNA clone 113nfectivity assay of ORSV full-length cDNA clones 113omplete sequence of ORSV infectious cDNA clone, pORSV-7 114equence comparison of ORSV-7 and other ORSV isolates 115he translation start site of ORSV MP ORF 116iscussions 118eferences 121ables 126able 1. Primers used in this study 126able 2. Percentage of sequence identity (%) between ORSV-7 and other ORSV isolates 127igures 128ig. 1 Schematic representation of genome organization of ORSV. 128ig. 2 The infectivity assay of ORSV full-length cDNA clones in Nicotiana benthamiana protoplasts. 129ig. 3 The symptom produced by ORSV infectious cDNA clones in N. benthamiana plants. 130ig. 5 Complete sequence of infectious cDNA clone, pORSV-7, and its encoded amino acid sequences for each ORF. 134ig. 6 The sequence comparison of predicted MP translation start sites in different ORSV isolates. 135hapter V 137he movement determinants of Odontoglossum ringspot virus 137bstract 139ntroduction 140aterials and methods 143onstruction of chimeric mutants 143rotoplast preparation and inoculation, Northern and western blot analysis of inoculated plants 143esults 144ifferential movement abilities of infectious cDNA clones pORSV-2 and -7 144he nucleotide and amino acid changes between pORSV-7 and -2 145he determinants of ORSV cell-to-cell movement 146he determinants of ORSV long-distance movement 148iscussions 152eferences 155igures 160ig.1 Schematic representation of the ORSV genome of infectious cDNA clones, pORSV-2, pORSV-7, and derivative constructions. 161ig. 2 The infectivity of ORSV infectious cDNA clones in N. benthamiana. 162ig. 3 Symptoms induced by ORSV wild type and infectious cDNA clones in local lesion and systemic hosts. 163ig. 4 The infectivity of ORSV infectious cDNA clones and their derivatives in N. benthamiana protoplasts. 164ig. 5 Symptoms induced by ORSV infectious cDNA clones and their derivatives in local lesion C. quinoa. 165ig. 6 The CP of ORSV was dispensable for it cell-to-cell movement. 166ig. 7 The systemic movement of ORSV infectious cDNA clones and their derivatives in N. benthamiana plants. 168application/pdf3104988 bytesapplication/pdfen-US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orchidCymMVORSVvirus detectionfull-length infectious cDNA clonehttp://ntur.lib.ntu.edu.tw/bitstream/246246/181924/1/ntu-97-D91633003-1.pdf