百合與唐菖蒲為本國重要的經濟花卉作物。其生產，不可避免地，與其他經濟作物同樣面臨病蟲害之威脅。在百合與唐菖蒲的重要病害當中，病毒病害以其能引起重大損失及難以傳統方法防治等因素，向被列為影響其生產的主要限制因子之一，其中以Cucumoviruses和Potyviruses為主要病毒。植物病毒病害的傳統防治方法一向只能治標，而能治本的抗病育種，則由於天然的抗病基因取得不易，且耗時耗力而顯得緩不濟急。所幸近年來，結合病原衍生型抗病性(pathogen-derived resistance, PDR)概念與基因轉殖技術所建構之轉基因抗病性(transgenic resistance)，已被廣泛且有效地應用於多種植物以抵抗植物病毒之為害。本計畫擬針對感染百合與唐菖蒲的胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)與Potyviruses選殖適用的病毒基因或基因片段建構具轉基因抗性的百合與唐菖蒲以解決CMV與Potyviruses為害百合與唐菖蒲的問題。在過去十數年間，病毒的基因如鞘蛋白基因(coat protein gene, CP)，複製酵素基因(replicase gene)等，均已被實際應用於病原衍生型抗病理論之實踐，然而成效參差不一。早期的研究顯示，轉殖病毒鞘蛋白基因於植物可有效地達到抗病毒的目的；然而近來也有研究顯示病毒CP雖可在轉基因植物內大量表現卻不能提供有效的抗性者。在數種目前可行的方法中，RNA中介式轉基因抗病性(RNA-mediated transgenic resistance)因能提供幾近於免疫的超強抗病能力而被認為是達成此目標之最佳選項。RNA中介式轉基因抗病性之產生即是由轉錄後基因消寂作用(post-transcriptional gene silencing, PTGS；或稱為RNA silencing)的現象所導致。目前已知雙股形態的RNA (dsRNA)是植物體啟動PTGS的主要啟始因子。本計畫擬利用CMV和Potyviruses病毒的基因片段分別構築出一個具有inverted repeat sequences的載體，使其能在轉錄時形成雙股核醣核酸(dsRNA)錄本，進而啟動PTGS機轉使轉基因百合或唐菖蒲得以表現RNA中介式轉基因抗病性而達到抗病毒的目的。本計畫之執行，預期將能產生抗CMV或抗Potyviruses的轉基因百合或唐菖蒲，將有助於本國之花卉產業，亦將有助於提升我國花卉種苗在國際市場銷售的競爭力。Lily and gladiolus are important economic ornamental crops to our floral industry. However, the production of lily and gladiolus have been suffered from the infection by several pests and diseases, including diseases caused by viruses. Among those lily- and gladiolus-infecting viruses, Cucumoviruses and Potyviruses are the major notorious viral agents. The establishment of resistance in plants through conventional plant breeding is one of the major ways for plant disease control. However, it is not a suitable one for viruses not only for lack of natural resistance genes but also being laborious and time-consuming. Alternative approaches, based on the concept of pathogen-derived resistance (PDR), such as transgenic expression of viral coat protein (CP) or (defective) replicase in plants to confer resistance against virus infection have been reported over the years, albeit with rather differing outcomes. Transgenic virus resistance can be engineered into plant genetically by using either functional virus genes or dysfunctional viral genomic fragments to provoke resistance at protein-level (protein-mediated) or at RNA level (RNA-mediated resistance). Among the various strategies that have been applied, RNA-mediated transgenic resistance has aroused as a promising one because of the extreme resistance it conferred. Plants showing RNA-mediated resistance were either completely resistant to virus infection or recovered from the initial infection. RNA-mediated resistance can be easily obtained by introducing constructs that formed double-stranded RNA (dsRNA) during the transcription of the transgenes. It is known that dsRNA forms are the key triggers of RNA silencing mechanisms. During induction of RNA silencing mechanisms, short small interfering RNAs (siRNAs) are generated from dsRNA by digestion with an RNaseIII-like enzyme (DICER). The siRNAs are proposed to subsequently guide an endonuclease complex (RISC) to RNA that shares sequence similarity with the inducing dsRNA and ensures that this RNA is specifically degraded. However, RNA-mediated viral resistance is a highly sequence specific process in which only RNA having homology to the transgenic inducer RNA is specifically degraded. This project specifically focuses on the establishment of transgenic resistance against CMV and potyviruses in lily and gladiolus by using inverted repeat constructs derived from CMV or potyvirus genomic fragments to provoke RNA silencing and, thus, transgenic resistance
