抗喜姆比蘭嵌紋病毒與齒舌蘭輪斑病毒之基因默化轉殖菸草抗病效力分析

Abstract

摘要 為了分析默化蘭花病毒鞘蛋白基因的效果，本研究選用穩定轉殖喜姆 比蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)或齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)全長與短片段鞘蛋白所構築之各默化 載體的邊沁菸草(Nicotiana benthamiana)，並以感病病葉萃取汁液直接接 種、Potato virus X (PVX)重組病毒與infectious clone三種方法分別進行接 種，記錄外觀病徵並以反轉錄聚合酶連鎖反應檢測植株中病毒鞘蛋白RNA 產物累積，檢測現有默化轉殖株的默化能力。試驗結果顯示三種病毒接種 方法皆可以將CymMV或ORSV順利的感染未轉殖之邊沁菸草，產生可辨識 的病徵，且會隨著時間增加趨於明顯。同時也可以在接種約一到兩週後的 葉片組織中，測得CymMV或ORSV鞘蛋白RNA的累積。CymMV或ORSV鞘 蛋白默化效果分析結果顯示：全長鞘蛋白RNAi及鞘蛋白短片段RNAi構築 之轉殖株，經接種後大部份轉殖株都表現出較輕微的病徵，同時也有較少 的病毒鞘蛋白mRNA產物累積。

To evaluate the silencing effect on Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) coat protein gene silenced construction, infected leaf sap, recombinant-Potato virus X (PVX) and virus infectious clone were used to inoculate Nicotiana benthamiana transformants containing different hairpin RNA (hpRNA) construct of RNA interference. Observation of virusderived symptoms and analysis by reverse transcription polymerase chain reaction (RT-PCR) were performed for investigation of silencing effect in transgenic lines. Untransformed N. benthamiana could be inoculated successfully with CymMV and ORSV by all three inoculation methods based on virusinduced symptoms appearance and accumulation of coat protein mRNA. After inoculation, transgenic lines containing hpRNA construct for coding region or 25 bp of coat protein cDNA form CymMV or ORSV showed milder virus-induceed symptoms and accumulated lesser coat protein RNA than untransformed N. benthamiana. These results confirmed the silencing effect of coat protein genes by hpRNA construct.