Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31354
標題: 以siRNA誘導抗二種馬鈴薯Y群病毒之轉基因植物之建構與帶有區分性突變點之馬鈴薯Y群病毒基因沉寂抑制子在病原性及病徵發展之分析
Generation of transgenic plants conferring siRNA-mediated resistance against two potyviruses, and analysis of discriminating mutations of potyviral gene-silencing suppressor on pathogenicity and symptom development
作者: 吳惠雯
Wu, Hui-Wen
關鍵字: ZYMV
矮南瓜黃化嵌紋病毒
PRSV W
improved cotyledon-cutting method
transgenic oriental melon
PTGS
siRNA
miRNA
木瓜輪點病毒西瓜型
改良式子葉切割法
轉基因東方甜瓜
後轉錄基因沉寂現象
小分子干擾核醣核酸
微小核醣核酸
出版社: 植物病理學系所
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摘要: 東方甜瓜的栽植遍佈全世界,在田間時常受到二種馬鈴薯Y群病毒的危害,分別為矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV) 及木瓜輪點病毒西瓜型 (Papaya ring spot virus type watermelon, PRSV W) 。 為了能有效的防治此二病毒,本研究在寄主方面,以遺傳工程技術建立簡單高效率的甜瓜轉殖方法以發展轉基因雙重抗病毒之甜瓜,結果顯示轉基因東方甜瓜的抗性來自於後轉錄基因沉寂現象 (Post-transcriptional gene silencing, PTGS)。此外,為了解馬鈴薯Y群病毒如何反擊植物防禦機制 (基因沉寂現象),進一步研究病毒ZYMV的基因沉寂抑制子 (gene silencing suppressor)-協同性蛋白 (Helper-component protease, HC-Pro) 如何影響小各種分子核醣核酸 (small RNAs) 所誘導的基因沉寂 (gene silencing),進而解釋病毒感染在植物上的病徵及病原性發展。本論文總共分為五個章節 (含附錄) , 分述如下。 本論文第一章前人研究,主要蒐集整理近幾年與本研究相關之參考文獻並概述本論文之目的與內容。 第二章至第四章為本論文主要架構,本研究欲利用育成轉基因抗病毒之植物來解決馬鈴薯Y群病毒在瓜類作物上所造成的損失。為了有效率的建構轉基因抗病毒之東方甜瓜,首先本研究發展一個操作簡單及高效率的改良式子葉切割法為甜瓜轉殖方法 (第二章),結果發現切除東方甜瓜子葉近軸端關鍵的 1 mm 部分可以減低偽陽性轉殖株的機率,進而提升成功轉殖的效率。本章以ZYMV CP為轉殖的基因,以期獲得抗ZYMV的轉基因東方甜瓜。結果顯示本研究獲得之ZYMV高抗或免疫的轉基因株系之抗性來自於基因沉寂機制,且其轉基因插入套數與抗性不完全相關。為了解決馬鈴薯Y群病毒在東方甜瓜上田間複合感染的問題,同樣的策略也應用在雙重抗ZYMV及PRSV W的轉基因東方甜瓜 (第三章),結果顯示高度抗病的轉基因東方甜瓜株系抗性來自於基因沉寂誘導抗性,其抗性程度與轉基因插入套數亦無完全相關。上述的單抗及雙抗的轉基因東方甜瓜將來可與其他田間危害嚴重之病毒鞘蛋白轉基因抗病株系進行雜交,在田間更有效率達到多重抗病毒之功效。此外,馬鈴薯Y屬病毒的協同性蛋白 (ptyviral HC-Pro) 是第一個發現的基因沉寂抑制子 (gene silencing suppressor),具有抑制基因沉寂的能力。本文第四章利用野生型及數個突變的HC-Pro轉入阿拉伯芥中,以阿拉伯芥呈現的表現型及分子證據去證實具不同突變點[R180I (A突變), F205L (B), and E396N (C)]組合的HC-Pro所造成的輕微及恢復性病徵,是由於其抑制了與病徵和病原性發展相關的miRNA,ta-siRNA及VIGS的能力受損所致,但這些突變的HC-Pro仍然保有抑制s-PTGS的能力。這些結果解釋也符合先前弱系病毒ZGAC (帶AC突變HC-Pro的ZYMV)產生輕微的病徵是由於其HC-Pro抑制miRNA及ta-siRNA pathway的能力降低所致,其恢復性病徵 (recovery)是因為其抑制能力與植物防禦反應維持一種平衡,故能發展成為提供良好交互保護的保護病毒 (protective virus)。上述研究基礎建立於後轉錄基因沉寂所誘導的抗性及基因沉寂抑制子如何藉由抑制不同小分子RNA影響病毒所引起之病徵及病原性,這些研究結果無論是對於利用轉基因或交互保護之策略來防治馬鈴薯Y群病毒,都能有更深入的了解,期望將來能夠解決在馬鈴薯Y群病毒田間危害嚴重的問題。 第五章為附錄 : “在人工微小核醣核酸誘導基因沉寂的環境下病毒非轉錄序列之分子演化” 。本研究於博士班在學期間 (2006-2008) 於美國洛克斐勒大學蔡南海院士實驗室完成,與林詩舜博士共列第一作者,非屬本論文主要架構,故收錄於附錄。此研究成果已於 2009 年二月正式刊登在 “PLoS Pathogens” 電子期刊 [PLoS Pathogens 5:e1000312] 。本章的研究目的為確立 21-nt 的人工病毒 miRNA 標靶區域 (miRNA target site) 對於 amiRNA (artificial microRNA) 轉基因植物所提供的抗性並進一步探討所有amiRNA 標靶區域上的單一核甘酸對amiRNA 結合的重要位置,結果發現病毒在轉基因表現amiRNA的壓力下會以剔除或突變核甘酸的方式逃避攻擊,本研究結果將是未來amiRNA轉基因抗病毒策略之重要設計參考。
Production of oriental melon (Cucumis melo L.) worldwide is often limited by infection by two potyviruses, the watermelon infecting Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus W type (PRSV W) and. In order to engineer melon lines resistant to these potyviruses, a construct containing the coat protein (CP) sequences of these viruses was generated and used to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Altogether, our results indicated that RNA-mediated post-transcriptional gene silencing (PTGS) was the underlying mechanism of virus resistance of the transgenic melon lines. In order to further understand the relationship between plant defense and virus counteraction, we investigated the role of gene silencing suppressor, potyviral HC-Pro, which affects small RNA pathways involving in pathogenicity and symptom development. This dissertation is divided into five (including appendix) chapters as described below. Chapter 1, “Literature review” describes references relevant to this study. Chapter 2, 3 and 4 comprise the main text of this thesis, describing the engineering of transgenic melon lines resistant to ZYMV and PRSV W (Chapter 2 and 3) to solve the potyvirus problem in the field. We used binary vector harboring single (ZYMV) or double (ZYMV and PRSV W) CP constructs to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Removal of 1 mm portion from the proximal end of cotyledon greatly increased the frequency of transgenic regenerants by significantly decreasing the incidence of false positive and aberrant transformants. Southern hybridization analysis of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants. Northern hybridization analysis evidenced an inverse correlation of the levels of accumulation of transgene transcript to the degrees of virus resistance, indicating post-transcriptional gene silencing (PTGS)-mediated transgenic resistance. Chapter 4 describes “Discriminating mutations of HC-Pro of Zucchini yellow mosaic virus with differential effects on small RNA pathways involving viral pathogenicity and symptom development”. It is well known that the potyviral HC-Pro is a gene silencing suppressor. We sought to obtain molecular evidence on the roles of the three highly conserved amino acids, R180I (mutation A), F205L (B), and E396N (C) of HC-Pro in microRNA (miRNA) and small interfering RNA (siRNA) pathways related to viral pathogenicity and symptom development using transgenic Arabidopsis plants system. We demonstrated that amino acid residues 180, 205 and 396 of HC-Pro are critical in suppression of miRNA, trans-acting siRNA (ta-siRNA) and virus induced gene silencing (VIGS) pathways, but not sense-post transcriptional gene silencing (s-PTGS). Because the R180I/ E396N HC-Pro mutant does not interfere with miRNA and tasiRNA pathways the ZGAC mutant elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the double AC mutations of HC-Pro. The findings of this study are useful to protect high levels of transgene expression and to genereate an attenuated potyvirus for control of virus by cross protection.. Chapter 5 (Appendix) entitled “Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing” was carried out at Prof. Nam-Hai Chua's laboratory, Rockefeller University, during my Ph.D program (2006-2008). The published paper is included as an appendix, because all the experiments of this project were carried out at Prof. Chua's laboratory and this study was not directly connected to thesis. The main objective of this chaper was directed to determine, through artificial mutagnesis, the criticality of single nucleotide positions in the amiRNA-targeted sequence of 21 nucleotides, which were constructed in a potyvirual vector. Furthermore, the evolution of the virus through deletion and substitution in amiRNA-targrted sequence to escape amiRNA recognition was also investigated.
URI: http://hdl.handle.net/11455/31354
其他識別: U0005-2605200912585200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2605200912585200
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