Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31167
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dc.contributor葉錫東zh_TW
dc.contributor詹富智zh_TW
dc.contributor張清安zh_TW
dc.contributor林長平zh_TW
dc.contributor.advisor陳煜焜zh_TW
dc.contributor.author陳德旭zh_TW
dc.contributor.authorChen, De-Shiuen_US
dc.contributor.other中興大學zh_TW
dc.date2009zh_TW
dc.date.accessioned2014-06-06T07:41:14Z-
dc.date.available2014-06-06T07:41:14Z-
dc.identifierU0005-0608200822262300zh_TW
dc.identifier.citation林學詩、全中和‧2000‧冬瓜新品種「花蓮1號」(吉豐)之育成及其特性‧花蓮區研究彙報 18:1-13。 Alonso-Prados J.L., Fraile A. and Garcia-Arenal F. 1997. Impact of cucumber mosaic virus and watermelon mosaic virus 2 infection on melon production in central Spain. J. Plant Pathol. 79: 131–134. An Y.Q., McDowell J.M., Huang S., McKinney E.C., Chambliss S. and Meagher R.B. 1996. Strong, constitutive expression of Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J. 10: 107-121. Anderson J.M., Palukaitis P., and Zaitlin M. 1992. A defective replicase gene induces resistance to Cucumber mosaic virus in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 89: 8759-8763. Baulcombe D.C. 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. The Plant Cell 8: 1833-1844. Bendahmane M., Chen I., Asurmendi S., Bazzini A.A., Szecsi J. and Beachy R.N. 2007. 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dc.identifier.urihttp://hdl.handle.net/11455/31167-
dc.description.abstract胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV) 是一種對經濟作物危害甚大的植物病毒,並在許多國家中造成了重大的經濟損失。針對胡瓜嵌紋病毒的危害有許多的防治策略,包含了抗病育種及建構轉基因抗性。與傳統抗病育種相較,建構轉基因抗性所需時間較少,因而成為現今在植物上建構病毒抗性的主流策略。胡瓜嵌紋病毒含有三條主要的核糖核酸,其上大部分的基因都有被直接應用於獲取轉基因抗性的實例,做為後轉錄基因默化抑制子的 2b 基因雖有應用部分序列以人造 miRNA 在轉基因植物中表現的方式構築抗性,卻未有直接應用全長度基因建構抗性的研究。蕃茄的胡瓜嵌紋病毒分離株 (CMV-peets) 藉由簡併式引子對選殖 2b 基因,經與已知的CMV 2b基因的核酸序列比對,可知 CMV-peets 的 2b 基因序列與屬於 IB 亞群的 CMV 之 2b 基因序列有極高的相同度。將 CMV-peets 2b 基因的轉譯架構導入蛋白質表現載體,透過大腸桿菌 (Escherichia coli) 表現出 2b 重組蛋白。以純化的 CMV 2b 重組蛋白製備抗血清,所產生的抗血清可應用於西方轉漬法以偵測同源抗原,但未能於生體內偵測到 CMV-peets 2b 蛋白。另一方面, 構築 5 種包含 2b 基因在內的轉基因構築,包括正向與反向的 CMV 2b ORF (pCMV2b-S 和 pCMV2b-AS)、正向與反向的 CMV 2b ORF 及兩側翼序列 (pCMV2b-EXT-S 和 pCMV2b-EXT-AS)、和 CMV 2b ORF 的反向重複序列 (pCMV2b-IR)。藉由農桿菌 (Agrobacterium tumefaciens) 導入模式植物菸草 (Nicotiana benthamiana) 中以測試應用 CMV 2b 基因建構病毒抗性之可行性。實驗結果顯示,五種構築中 pCMV2b-IR 在 R0 轉基因植株提供最佳的抗病性,其餘四種單一基因構築的載體亦可提供抗性於轉基因植株。zh_TW
dc.description.abstractCucumber mosaic virus (CMV) causes severe damage and economical loss to many commercial crops. Many approaches, including conventional resistance breeding and transgenic resistance, have been developed to confer resistance in plants against Cucumber mosaic virus. Comparing to the time-consuming conventional measures, transgenic approaches are currently the main stream of building resistance against viruses in plants. CMV contains three major RNAs, most genes on these RNAs have been applied to generate transgenic resistance in the literature. The 2b gene has applied to generate transgenic resistance with expressing artificial RNA in plants, however, never applied in the form of full length. In this study, the 2b gene of a tomato isolate of CMV (CMV-peets) was amplified with a degenerated primer pair and the product was cloned. Sequence analysis showed that the nucleotide of the 2b gene of CMV-peets is highly homologous to that of CMV isolates belong to subgroup IB. The 2b open reading frame was introduced into protein expression vector to express 2b recombinant protein in E.coli. The expressed CMV 2b protein was purified and applied to produce a polyclonal antiserum. The produced antiserum was applicable to detect homologous antigen in vitro, but failed to detect CMV 2b protein for immunoblot tests. In the mean time, five constructs consist of 2b ORF in sense or anti-sense orientations (pCMV2b-S and pCMV2b-AS, respectively), 2b ORF with flanking sequences in sense or anti-sense orientations (pCMV2b-EXT-S and pCMV2b-EXT-AS, respectively), and 2b ORF in inverted repeat form (pCMV2b-IR), were transferred into tobacco (Nicotiana benthamiana) mediated by Agrobacterium tumefaciens. Our results indicated all five constructs confer transgenic resistance to plants and the inverted repeat construct has the best efficiency among the five constructs.en_US
dc.description.tableofcontents中文摘要……………………………………………I 英文摘要……………………………………………II 前言…………………………………………………1 前人研究……………………………………………4 材料與方法…………………………………………11 病毒來源與保存………………………………11 總量 RNA 之抽取………………………………11 引子對之設計…………………………………11 CMV 2b基因選殖………………………………12 CMV 2b基因於蛋白質表現載體(pET28)中之 表現及CMV 2b基因產物之純化………………13 抗血清之製備…………………………………14 蛋白質膠體電泳分析(sodium dodecyl-sulfate polyacrylamide gel electrophoresis, SDS-PAGE)………………………………………14 西方轉漬法 (western blotting)……………15 CMV 2b 轉基因載體之構築……………………16 農桿菌(Agrobacterium tumefaciens LBA4404) 之轉型……………………………………………17 轉型農桿菌之篩選 (Selection of transformed Agrobacterium tumefaciens)…………………17 轉基因菸草之轉殖與再生………………………18 轉殖株抗性測試…………………………………19 轉殖株DNA之萃取…………………………………19 轉殖株PCR (polymerase chain reaction) 檢測…………………………………………………20 結果………………………………………………………21 CMV 2b基因之選殖…………………………………21 CMV 2b 基因序列分析……………………………21 CMV 2b 基因導入蛋白質表現載體 (pET28)………21 CMV 2b 蛋白表現及純化…………………………22 CMV 接種菸草(Nicotiana benthamiana) 與奎藜(Chenopodium quinoa)……………………22 轉基因植株之抗性表現……………………………23 轉基因植株病徵觀察………………………………23 討論………………………………………………………25 參考文獻…………………………………………………29 圖表………………………………………………………39 附錄………………………………………………………63zh_TW
dc.language.isoen_USzh_TW
dc.publisher植物病理學系所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0608200822262300en_US
dc.subjectCMVen_US
dc.subject胡瓜嵌紋病毒zh_TW
dc.subjecttransgenic resistanceen_US
dc.subjectantiserumen_US
dc.subject轉基因抗性zh_TW
dc.subject血清zh_TW
dc.title應用胡瓜嵌紋病毒2b基因建構轉基因抗性及2b基因產物製備專一性抗血清zh_TW
dc.titleApplying Cucumber mosaic virus 2b gene to construct transgenic resistance and producing specific antiserum against 2b proteinen_US
dc.typeThesis and Dissertationzh_TW
Appears in Collections:植物病理學系
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