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研究生:施傑仁
研究生(外文):Jie-Ren Shih
論文名稱:利用基因消寂機制發展抗多種病毒病害之轉基因茄科植物
論文名稱(外文):Development of transgenic solanaceous plants resistant to multiple viruses via gene silencing
指導教授:詹富智
指導教授(外文):Fuh-Jyh Jan
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:62
中文關鍵詞:基因消寂機制轉基因植物西瓜銀斑病毒蕃茄斑萎病毒屬多重抗病轉錄後基因消寂機制
外文關鍵詞:gene silencingtransgenic plantWSMoVtospovirusmultiple resistancePTGS
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中文摘要
1985年Sanford和Johnston提出了pathogen-derived resistance (PDR) 的概念,認為於轉基因植物中表現病原的基因可以提供對該病原或具有同源性或相關病原的抗性,也是轉基因植物抗病毒病害的理論基礎。 先前詹老師等人於美國的研究結果發現,將400 bp的Tomato spotted wilt virus (TSWV) 核鞘蛋白基因 (N gene) 片段連結三種病毒: TSWV, Groundnut ringspot virus (GRSV) 及Impatiens necrotic spot virus (INSV) 的核鞘蛋白基因片段200bp轉入煙草 (Nicotiana benthamiana) 中,發現可產生對此三種病毒的抗性。 根據這些發現,詹老師等人提出假說認為將不同病毒的小片段基因連接至大約 400 bp 的基因片段 (silencer DNA) 時可誘導植物的基因消寂機制並得到具多種病毒抗性的轉基因植物。 於是本實驗選用了台灣本土茄科和葫蘆科上的重要病毒病害 - 西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 的 N 基因片段 (m/2Nw, 378 bp) 作為silencer DNA,藉以誘發植物產生多種病毒抗性。 結果在轉入m/2Nw 基因連結長度為200 bp的TSWV, GRSV及INSV核鞘蛋白基因片段時,有些轉基因煙草株系 (lines) 可以同時產生對TSWV, GRSV, INSV三種病毒的抗性。 此外,我們亦探討此一策略應用於不同屬間的病毒及病毒鞘蛋白以外基因之可行性,Potato virus Y (PVY), Cucumber mosaic virus (CMV) 和 Tomato mosaic virus (ToMV) 分別屬於不同的病毒屬,將它們的鞘蛋白基因及 ToMV 之複製酶 ( replicase) 基因片段各約250 bp,每三個片段為一組連結於 m/2Nw 基因片段之後產生 m/2Nw-CToY, m/2Nw-CRY 和m/2Nw-YCTo 三個轉殖載體,用於進行煙草基因轉殖及研究其抗病性。 目前R0 代煙草在接種 PVY 後已得到三個抗 PVY 之品系,未來將於R1子代繼續分析對於PVY,CMV及ToMV之抗性。
Abstract
The concept of pathogen-derived resistance (PDR) postulated by Sandford and Johnston states that plants transformed with genes from pathogen may confer resistance to homologous or related pathogens. Previous studies of Jan et al. had shown that N gene segments (400 bp) of Tomato spotted wilt virus (TSWV) fused with three shorter N gene segments (200 bp) of TSWV, Groundnut ringspot virus (GRSV), and Impatiens necrotic spot virus (INSV) can induce PTGS by developing resistance against TSWV, GRSV, and INSV in Nicotiana benthamiana plants.
Based on these results, Jan et al. formulated a hypothesis stating that transforming plants with transgene constructs having gene segments of different viruses linked to a universal silencer DNA can produce multiple virus resistance. In this study, we transformed N gene segment of Watermelon silver mottle virus (WSMoV) fused with three shorter N gene segments of TSWV, GRSV, and INSV to N. benthamiana plants. The results showed that transgenic plants with this construction can confer resistance to these three tospoviruses. To generate multiple resistance among viruses belonging to different genus and to confirm the use of the genes other then CP gene, nearly 250 bp CP gene segments of Potato virus Y (PVY), Tomato mosaic virus (ToMV), Cucumber mosaic virus (CMV), and segment of ToMV replicase gene were fused with WSMoV N gene segment and transformed to N. benthamiana plants. The R0 resistant lines obtained were challenged with PVY and three lines have shown high level of resistance. The seedlings collected from these will be subjected to select multiple viral resistance progenies.
Contents
中文摘要…........... 3
Abstract in English………… 4
前言........................... 5
Introduction………….......… 12
Materials and Methods
Virus source…………...………...…….. 17
Isolation of total RNA……………………17
Construction of plant expression and transformation vectors containing middle half N gene of WSMoV as a silencer DNA and linked with triple 1/4 N gene segments from three Tospovirus…...........................18
Construction of plant expression and transformation vectors containing triple 1/4 CP gene segments from different group of viruses linked to a silencer DNA...........19
Tobacco transformation………………….. 19
Tomato transformation20
DNA extraction from plant tissue…………………….21
PCR detection of the transgenes in transgenic plants…… 22
Indirect-ELISA test…………………………………….22
Selection of R0 resistance transgenic lines by challenging virus....23
Segregation and multiple virus resistance analysis of R1 progeny...... 24
Northern blot analysis...…………………………... 25
Results
Construction of plant expression and plant transformation vector…..… 26
Tobacco transformation…………………………..…………….… 28
Tomato transformation………………………….……………...… 29
Screening of m/2Nw gene transformed R0 lines resistant to WSMoV …30
Screening of GRSV resistant m/2Nw-ITG transformed R0 lines… 31
Screening of PVY resistance transgenic tobacco R0 plants expressing m/2Nw-CToY, m/2Nw-YCTo and m/2Nw-CRY........ 32
Segregation and multiple virus resistance of transgenes in R1 progeny…………..... 32
Northern hybridization for detecting the expression level of transgenes…………………………………………………………... 34
Discussion…………………………………………………………………. 35
References…………………………………………………………………. 40
Figures and Tables……………….…... 48
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