建構轉基因抗性以抗洋桔梗壞疽病毒之可能性探討

Preliminary studies on constructing transgenic resistance against Lisianthus necrosis virus

"洋桔梗（Eustoma russellianum（Don.）Griseb）為重要的切花類觀賞花卉，在洋桔梗的栽培過程中常受到病毒病害的感染，其中以洋桔梗壞疽病毒（Lisianthus necrosis virus, LNV）所造成的危害最具威脅性。本研究探討使用後轉錄基因靜默機制（post-transcriptional gene silencing, PTGS）誘導植物產生抗LNV之轉基因抗性之可能。針對LNV的基因體中RdRP、CP、MP等基因設計了四個目標區域，以反向重複序列的方式構築於二元載體pBIN（pLNVRD5-IR、pLNVRD3-IR、pLNV3'CP-IR、pLNVMP-IR），利用農桿菌轉殖法（Agrobacterium tumefaciensmediated transformation, ATMT）將其導入模式植物之圓葉菸草（Nicotiana benthamiana）。菸草R0轉殖株的結果LNV RdRP基因的前半（5'端）所構築的反向重複序列（pLNVRD5-IR）所提供的轉基因抗性為最高，可達到80.1%，其餘三種反向重複序列構築-RdRP基因的後半（3'端）（pLNVRD3-IR）、外鞘蛋白基因的後半（3'端）（pLNV3'CP-IR）、與移動蛋白基因（pLNVMP-IR）亦可使轉殖株產生對LNV之抗性，抗病比率依序分別為32%、41.2%與12.5%。"

"Lisianthus [Eustoma russellianum (Don.) Griseb.] is an important ornamental crop in the cut flower industry worldwide. However, there are several viral diseases interfere lisianthus production in Taiwan, especially Lisianthus necrosis virus (LNV), which is probably the most devastating one to lisianthus. In order to provide lisianthus with virus resistance, the effective strategies of post-transcriptional gene silencing (PTGS) was chosen. Among those, the approach of using inverted-repeat (IR) constructs has been shown to have better efficacy. The cDNA fragments derived from LNV genes of RNA-dependent RNA polymerase (RdRp), coat protein (CP) and movement protein (MP) were cloned into the binary vector pBIN in the form of IR and already introduced into Nicotiana benthamiana and Eustoma russellianum by Agrobacterium tumefaciens-mediated transformation (ATMT) to generate transgenic tobacco and lisianthus potentially resistant to LNV. All four IR constructs, i.e. pLNVRD5-IR, pLNVRD3-IR, pLNV3'CP-IR, and pLNVMP-IR, conferred transgenic resistance to tobacco plants and the pLNVRD5-IR conferred the best efficiency among the four constructs. The resistance ratio in R0 tobacco of constructs pLNVRD5-IR, pLNVRD3-IR, pLNV3'CP-IR and pLNVMP-IR are 80.1%, 32%, 41.2% and 12.5%, respectively."