蘭花抗病毒專一性載體之功能分析與文心蘭基因轉殖之研究

Abstract

摘要 蘭花是台灣重要的園藝作物之ㄧ，本論文藉由基因轉殖，導入文心蘭乙烯受體突變基因ers1，以降低對乙烯敏感性與延長瓶插壽命；以核醣核酸干擾技術產生具有默化齒舌蘭輪點病毒(Odontoglossum ringspot virus; ORSV)與喜姆比蘭嵌紋病毒(Cymbidium mosaic virus ;CyMV)能力之植株；導入果膠分解酶(pectate lyase ; PL)基因之ㄧPelE-1基因，提升文心蘭植株對軟腐病之抗病能力。 由RNA干擾使mRNA發生降解，作為植物抗病毒之策略。本試驗以農桿菌轉殖法，轉殖ORSV與CyMV全長與短片段外鞘蛋白所構築之各默化載體，穩定轉殖到菸草(Nicotiana benthamiana)。菸草轉殖株經過GUS活性組織化學染色與聚合酶連鎖反應(polymerase chain reaction ; PCR) 分析，各構築均獲得轉殖株，將轉殖株分別接種ORSV與CyMV病毒，抽取病葉RNA，以反轉錄聚合酶連鎖反應(reverse transcription polymerase chain reaction ; RT-PCR)分析後得知可能具有默化CyMV核酸能力的轉殖系為pGCR4-5-4，具有默化ORSV核酸能力的轉殖系為pGOR4-1-2、pGOR4-2與pGORA18，這幾個轉殖系偵測不到病毒外鞘蛋白基因，推測於菸草的體內默化機制已啟動。 以文心蘭‘Gower Ramsey’癒傷組織為材料，藉由農桿菌媒介法進行基因轉殖。將自文心蘭得到之乙烯受體基因OgERS1進行點突變後，利用農桿菌轉殖法導入文心蘭中，以G418篩選後，細胞與PLB分別以GUS染色，已獲得75擬轉殖系，再將其中12個轉殖系細胞，抽取DNA進行PCR分析，有5個轉殖系增幅得到1342 bp長的片段，證明ers1導入文心蘭中。Erwinia chrysanthmi感染植物後會分泌PL，此酵素將細胞壁之果膠質分解成小分子的OG (oligogalacturonates)，OG可誘導植物產生抗病反應，將PelE導入文心蘭，已獲得28個擬轉殖系，期望可以得到抗軟腐病之植株。目前栽培的文心蘭切花品種大多受到病毒感染，本論文亦期望藉由基因默化的策略得到抗CyMV以及ORSV之文心蘭轉殖株。將ORSV全長外鞘蛋白RNA干擾構築以農桿菌法轉殖文心蘭癒傷組織，以篩選標誌GFP檢測有7個細胞系可以在500-560 nm的波長下被激發出綠色螢光，證明轉殖事件的發生。

Abstract Orchids are the most important horticultural crop in Taiwan. In this study, a mutated ethylene receptor gene (Og-ERS1) from Oncidium for generation of transgenic Oncidium with reduced ethylene sensitivity and extension of the vase life of the flowers. We also generated transgenic plants with the ability to degrade the RNA of Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CyMV) by RNA interference (RNAi). In order to enhance resistance to the soft-rot disease, pectate lyase gene PelE-1 isolated from Erwinia chysanthemi was transformed into callus of Oncidium. RNAi is an effective strategy for engineering plants with resistance to generate virus-resistance transgenic plants. In this study, a full length and four conserved regions of ORSV coat protein (CP) and a CyMV CP to Nicotiana benthamiana, by Agrobacterium tumefaciens as a tool. Putative transgenic tobaccos were further confirmed by GUS histochemical staining. and polymerase chain reaction (PCR) analyses. Inoculaion assay revelved that trangsenic line pGCR4-5-4 resistance to CyMV and line pGOR4-1-2, pGOR4-2 and pGORA18 resistance to ORSV infection. The results indicated CyMV and ORSV CP did not exist in four transgenic lines, pGCR4-5-4, pGOR4-1-2, pGOR4-2 and pGORA18, indicating the RNAi mechanism has already been started. Genetically transformed plants of Oncidium ‘Gower Ramsey’ were regenerated after cocultivation of callus with Agrobacterium tumefaciens. Oncidium ethylene receptor gene OgERS1 with missense mutation was introduced into Oncidium, and 75 transformants were surrived after G418 selection. Putative transgenic calli and PLBs were further assayed by GUS histochemical staining and polymerase chain reaction (PCR) analyses. A DNA fragment of ca. 1342-bp was observed in five putative transgenic lines, indicating the mutated OgERS1 was introduced into the genome. Pectate lyase (PL) secreted by Erwinia chrysanthmi de-polymerises pectin of plant cell wall into unsaturated oligogalacturonates (OG) that can trigger various plant defence responses. PelE-1, an isoenzyme of PL, was transferred to Oncidium callus using Agrobacterium tumefaciens as a tool, and expected to gain the soft-rot resistance. There were 28 transformants obtained, using G418 as a selection agent. Presently, most of Oncidium were infected by ORSV and CyMV. In this study, we also attempt to engineered plant that will resistance to ORSV. There are seven transformed callus lines screening by using GFP as a reporter.