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標題: 構築洋桔梗壞疽病毒之轉基因抗性
Construction of Transgenic Resistance against Lisianthus necrosis virus
作者: Yu-Wei Lin
關鍵字: 洋桔梗壞疽病毒
Lisianthus necrosis virus
transgenic resistance
inverted-repeat construct
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摘要: 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。為了使植物具備病毒抗性,目前已有傳統的抗病育種,以及建構轉基因抗性等方法。但抗病育種所需時間較長,相對而言轉基因抗性較為省時又能表現穩定專一的抗性,因此成為建構病毒抗性之主流。本研究選擇使用後轉錄基因靜默機制 (post-transcriptional gene silencing, PTGS)誘導洋桔梗產生病毒抗性。根據前人研究,不論將正向或反向的病毒序列導入植物體內皆能誘導siRNA產生進而引發PTGS,其中又以反向重複序列的構築方式能提供最佳的抗病效率。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 tumefaciens-mediated transformation, ATMT)將其導入模式植物圓葉菸草 (Nicotiana benthamiana)及洋桔梗 (Eustoma russellianum)中。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)、移動蛋白基因 (pLNVMP-IR)-亦可使轉殖株產生對LNV之抗性,抗病比率依序為32%、41.2%與12.5%。相同的構築已轉殖至洋桔梗,因轉殖株的生長速度較慢,目前仍在進行中,尚未進行抗病性的相關測試。
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. There were many approaches, including conventional resistance breeding and transgenic resistance, have been developed to build resistance in plants against viral diseases. Compared to the time-consuming conventional resistance breeding, transgenic approaches can provide more stable and specific resistance efficiency. In this study, in order to provide lisianthus with virus resistance, the effective strategy of post-transcriptional gene silencing (PTGS) was chosen. Based on previous researches, transgenic plants harboring viral sequences, both in sense or in anti-sense orientations, can trigger the formation of siRNA and thus lead to PTGS. These have been demonstrated successfully in several crops. Among these, 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 separately 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. The transformation and regeneration of lisianthus are now under processing. However, due to the limitation of growth rate of tissue-cultured lisianthus, the transgenic resistance of these transformants have not been analyzed yet.
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系



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