抗三種蝴蝶蘭病毒轉基因植物之研發

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抗三種蝴蝶蘭病毒轉基因植物之研發

Development of transgenic plants with multiple resistance to three Phalaenopsis orchids-infecting viruses

李佳華 , Masters　　Advisor：詹富智　　

繁體中文

蝴蝶蘭病毒；轉基因植物；多重病毒抗性； Phalaenopsis orchids-infecting virus ； transgenic plant ； multiple resistance

Abstract │ Reference (133) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

蝴蝶蘭 (Phalaenopsis spp.) 是台灣重要的外銷花卉作物，其多是以組織培養方式大量繁殖，在栽培過程中易遭受多種植物病原菌感染，其中又以病毒病害最難以防治。目前主要感染蝴蝶蘭之病毒為蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV)、齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV) 及番椒黃化病毒 (Capsicum chlorosis virus, CaCV-Ph) 三種，不僅引起嚴重病徵，還會影響產量及造成重大經濟損失，而且蝴蝶蘭栽培期間不只會被一種病毒感染，故為了解決蘭花病毒病害的問題，本研究針對此三種蝴蝶蘭病毒，運用植物基因工程技術及轉錄後基因沉寂 (post-transcriptional gene silencing, PTGS) 的策略，藉由轉基因表現連結多種病毒基因片段，發展同時具有多重蝴蝶蘭病毒抗性之轉基因植物。研究中選殖CymMV、ORSV及CaCV-Ph之複製酶部分基因片段，構築兩種帶有連結此三段基因的轉殖載體，一為傳統轉殖載體pCAMBIA1304-CyORCaRep，另一個則是可以產生無篩選標誌之載體pGA2TNH-CyORCaRep，並藉由農桿菌進行模式植物菸草 (Nicotiana benthamiana) 轉殖以及蝴蝶蘭轉殖，由於蝴蝶蘭轉殖與再生需要較長的時間，故先以模式植物分析構築的轉殖載體所誘發轉基因植物對病毒產生抗性的機制及效果，評估載體用於發展具有多重病毒抗性之轉基因蝴蝶蘭的可行性。轉殖再生的菸草株系分別以PCR與南方雜合反應確認轉殖成功的轉基因菸草，其中轉殖pCAMBIA1304-CyORCaRep獲得38株確定的轉基因株系，而轉殖pGA2TNH-CyORCaRep獲得7株帶有目標基因的轉殖株系，總共有45個株系挑戰接種病毒ORSV或CaCV-Ph，接種結果得到pCAMBIA1304-CyORCaRep有5株系為對ORSV具有抗病性的轉基因菸草，另外pGA2TNH-CyORCaRep有3株系具有對CaCV-Ph的抗性，以及1株系對CaCV-Ph延遲發病的轉基因株系，將這些抗病轉殖株自交後得到的R1子代，進一步以病毒接種分析子代的抗病性，結果證明此抗病能力能夠遺傳至後代。此外，本研究選用蝴蝶蘭V3大白花品種 (Phalaenopsis Sogo Yukidian 'V3') 之擬圓球體 (protocorm-like bodies, PLBs) 作為轉殖培植體，利用基因槍轉殖法及農桿菌轉殖法進行大量的蝴蝶蘭轉殖，目前以抗生素篩選後已得到103個可能轉殖株，其中6個經PCR及南方雜合反應確認帶有目標轉基因，後續待其增殖生長後即可進行病毒接種測試抗病性。此多重抗病的轉殖策略除了可應用於蝴蝶蘭親本抗病育種外，還能運用在其他蘭花，如石斛蘭、文心蘭，期望藉此可降低病毒病害對蘭花產業的為害。

Parallel Abstract 〈TOP〉

Phalaenopsis orchids (Phalaenopsis spp.) are commercially important export ornamental plants cultivated as cut flowers and potted plants in Taiwan. Currently, the Phalaenopsis orchids are commonly propagated by tissue culture technology, but the productivity are limited by various pathogens, especially the orchid-infecting viruses. Three viruses, including Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV) and Capsicum chlorosis virus (CaCV-Ph), were the most predominant viruses that infect Phalaenopsis orchids in Taiwan. The fact that orchids are frequently invaded by more than one virus during the breeding process prompted this study to develop transgenic plants with multiple resistance to three Phalaenopsis orchids-infecting viruses. The gene silencing strategy was used to generate transgenic plants by introducing the chimeric transgene containing the three viruses’ gene fragment. The individual partial-replicase genes of the three viruses was fused together and cloned into the expression vector. The expression cassette contained the chimeric transgene was introduced into two transformation vectors, one is the traditional vector, pCAMBIA1304 and the other is marker-free binary vector, pGA2TNH. The two constructs were transformed into Nicotiana benthamiana and Phalaenopsis orchids separately. In transformed N. benthamiana, 38 transgenic tobacco plant lines derived from pCAMBIA1304-CyORCaRep were obtained and the insertion of the transgene was confirmed by PCR and Southern blot. In the marker-free system, pGA2TNH-CyORCaRep, 7 transgenic tobacco plant lines carried the viral transgene. All 45 lines carried viral transgene were challenged with ORSV or CaCV-Ph to evaluate their resistance to respective virus. Results of the resistance assays showed 5 lines were resistant to ORSV, 3 lines to CaCV-Ph and 1 line with delayed symptom to CaCV-Ph. Seeds of the resistant lines were collected and used to evaluate the inheritance of viral resistance. Eighteen percent of the progeny plants were resistant to ORSV infection whereas 28% showed resistant to CaCV-Ph infection. In addition, these two constructs were transformed into protocorm-like bodies (PLBs) of Phalaenopsis Sogo Yukidian 'V3' by particle bombardment and Agrobacterium-mediated transformation. So far, 103 putative lines of transgenic Phalaenopsis orchid were screened from the selection medium, and 6 of them were confirmed the presence of transgene by PCR and Southern blot carried the target transgene. The transgenic orchid lines are being propagated in large scale and will be used in resistance evaluation. The orchids with multiple resistances developed in this study could be used for resistance breeding program and the constructs could be applied in other orchid cultivar such as Dendrobium and Oncidium orchids to control the virus disease in orchid production.

Reference ( 133 ) 〈TOP〉

江宜樺。2009。利用轉錄後基因沉寂發展具有多重病毒抗性之轉基因西瓜。國立中興大學農藝學系碩士論文。台中。
連結：
何琇銀。2007。抗胡瓜嵌紋、胡瓜綠斑嵌紋與西瓜嵌紋病毒轉基因西瓜之研發。國立中興大學農藝學系碩士論文。台中。
連結：
林靜宜。2010。抗台灣番茄捲葉病毒與番茄斑萎病毒無篩選標誌轉基因植物之研發。國立中興大學植物病理學系博士論文。台中。
連結：
Baker, C. A., Davison, D., and Jones, L. 2007. Impatiens necrotic spot virus and Tomato spotted wilt virus disgnosed in Phalaenopsis orchids from two Florida nurseries. Plant Dis. 91: 1515.
連結：
Baulcombe, D. 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8: 1833-1844.
連結：

Times Cited (1) 〈TOP〉

簡奕馨(2017)。奈米結構檢測晶片於齒舌蘭嵌紋病毒之快篩應用。中興大學機械工程學系所學位論文。2017。1-74。

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