Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89334
DC FieldValueLanguage
dc.contributorYuh-Kun Chenen_US
dc.contributor陳煜焜zh_TW
dc.contributor.authorYu-Wei Linen_US
dc.contributor.author林郁瑋zh_TW
dc.contributor.other植物病理學系所zh_TW
dc.date2015zh_TW
dc.date.accessioned2015-12-07T07:25:30Z-
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Plant Cell Tiss. Org. Cult. 113: 571-578.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/89334-
dc.description.abstract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。zh_TW
dc.description.abstractLisianthus (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.en_US
dc.description.tableofcontents中文摘要……………………………………………………………………………i Abstract…………………………………..…………………………………………ii 目次…………………………………..……………………………………………..iv 表目次…………………………………..…………………………………………..vi 圖目次…………………………………..…………………………………………..vii 壹、前言……………………………………………………………………………1 貳、前人研究………………………………………………………………………4 參、材料與方法 病毒接種源與抗血清來源…………………………………………………...16 病毒顆粒形態觀察與病組織超薄切片 (Ultrathin section)……………..….16 病毒純化…………………………………………………………………..….17 抗血清製備……………………………………………………………..…….17 植物基因組抽取 A、總量核糖核酸 (Total RNA)抽取……………………………….…18 B、基因體去氧核醣核酸 (Genomic DNA)抽取……………………...18 核酸分析 A、反轉錄聚合酶連鎖反應…………………………………………....18 B、聚合酶連鎖反應……………………………………………………19 目標基因之選殖………………………………………………………...…....20 反向重複序列 (inverted-repeat)轉基因載體之構築...…………….………..20 農桿菌轉型…………………………………………………………………...20 菸草轉型 (Transformation of Nicotiana benthamiana)………………….…..21 菸草轉殖株挑戰接種 (Challenge inoculation)……………………………...22 菸草轉植株F1子代篩選及挑戰接種………………………………………..22 間接酵素連結免疫吸附反應 (Indirect-enzyme-linked immunosorbent assay, Indirect-ELISA)………………………………………………………………22 北方轉漬法 (northern blotting)……………………………………………...23 洋桔梗轉型 (Transformation of Eustoma russellianum)…………………….24 肆、結果 LNV感染寄主之病徵觀察………………………………………………..…25 LNV顆粒形態與罹病組織超薄切片觀察…………………………………..25 LNV目標基因之選殖………………………………………………………..25 反向重複序列 (inverted-repeat)轉基因載體之構築………………………..26 農桿菌篩選…………………………………………………………………...27 菸草轉型……………………………………………………………………...27 轉基因菸草之病徵觀察……………………………………………………...27 轉基因菸草之抗病分析……………………………………………………...28 轉基因R0菸草siRNA偵測………………………………………………....29 轉基因菸草F1子代挑戰接種………………………………………………..29 洋桔梗轉型…………………………………………………………………...30 伍、討論…………………………………………………………………………...31 陸、參考文獻……………………………………………………………………...36 柒、表……………………………………………………………………………...47 捌、圖……………………………………………………………………………...50 表一、洋桔梗病毒病害列表………………………………………………………47 表二、本研究中所使用的引子對序列……………………………………………48 表三、轉基因R0圓葉菸草 (N. benthamiana)接種洋桔梗壞疽病毒 (LNV)之抗病 表現…………………………………………………………………………49 圖一、洋桔梗壞疽病毒 (LNV)感染洋桔梗造成的病徵……………………….…50 圖二、洋桔梗壞疽病毒(LNV)接種寄主植物之病徵……………………………...51 圖三、洋桔梗壞疽病毒 (LNV)之形態………………………………………….…52 圖四、洋桔梗壞疽病毒 (LNV)基因體簡圖與選殖策略………………………….53 圖五、使用專一性引子對以RT-PCR方式增幅出洋桔梗壞病毒 (LNV)上四個目標 基因片段……………………………………………………………………....54 圖六、以限制酶確認於pRAP載體上四個洋桔梗壞疽病毒 (LNV)之轉基因 構築…………………………………………………………………………..55 圖七、以洋桔梗壞疽病毒 (LNV)基因序列構築反向重複序列 (inverted-repeat, IR) 轉基因載體之示意圖…………………………………………………………56 圖八、菸草轉植株挑戰接種洋桔梗壞疽病毒(LNV)30天後之發病情形………..58 圖九、利用間接酵素連結免疫吸附反應 (Indirect-ELISA)檢測菸草轉殖株 (R0)挑 戰接種洋桔梗壞疽病毒 (LNV)後之病毒累積情形……………………….59 圖十、抗病的轉基因株系 (pLNVRD5-IR)之抗病反應與小片段干擾RNA (siRNA) 之檢測…………………………………………………………………………60 圖十一、抗病的轉基因株系 (pLNVRD5-IR)的F1子代挑戰接種洋桔梗壞疽病毒 (LNV) 10天後之抗病表現……………………………………………….61 圖十二、轉基因洋桔梗於kanamycin選擇性培養基上的生長情形……………...62zh_TW
dc.language.isozh_TWzh_TW
dc.rights不同意授權瀏覽/列印電子全文服務zh_TW
dc.subject洋桔梗壞疽病毒zh_TW
dc.subject轉基因抗性zh_TW
dc.subject反向重複序列構築zh_TW
dc.subject洋桔梗zh_TW
dc.subjectLisianthus necrosis virusen_US
dc.subjecttransgenic resistanceen_US
dc.subjectinverted-repeat constructen_US
dc.subjectlisianthusen_US
dc.title構築洋桔梗壞疽病毒之轉基因抗性zh_TW
dc.titleConstruction of Transgenic Resistance against Lisianthus necrosis virusen_US
dc.typeThesis and Dissertationen_US
dc.date.paperformatopenaccess2018-02-03zh_TW
dc.date.openaccess10000-01-01-
Appears in Collections:植物病理學系
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