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標題: 以單一轉基因誘發後轉錄基因沉寂作用發展抗多種病毒之轉基因東方甜瓜
Engineering multiple virus resistance based on post-transcriptional gene silencing mechanism in oriental sweet melon (Cucumis melo L.) using single chimeric gene constructs
作者: 潘維尼
Panwar, Vinay
關鍵字: RNA silencing
出版社: 植物病理學系所
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摘要: 自從大多數作物在田間漸漸趨向於數種病毒複合感染之危害,開發能夠提供重要經濟性作物多重病毒抗性的技術可以有效減少農業上的損失。葫蘆科作物,包含瓜類 (Cucumis melo L.) 在內,容易遭受許多不同的病毒危害造成嚴重損失。影響瓜類生產的三種主要病毒為矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus, ZYMV)、木瓜輪點病毒 W 型 (Papaya ringspot virus type watermelon, PRSV-W) 以及胡瓜嵌紋病毒 (Cucumber mosaic virus, CMV)。在葫蘆科作物中常常偵測到病毒複合感染致使在作物上為害加重的例子,因此利用基因工程策略來培育能抵抗多重病毒感染之抗性作物品種至為重要。本實驗的主旨是根據後轉錄基因沉寂(post-transcriptional gene silencing, PTGS )的理論,發展具有廣泛性抗性的轉基因甜瓜以對抗上述三種分別隸屬於兩個不同病毒族群的主要感染葫蘆科作物的病毒 (ZYMV, PRSV-W 和 CMV)。 在本研究中,我們構築一段易誘導基因沉寂的序列(silencer DNA) 融合上述三種病毒序列片段作為轉殖載體,以達成高效率多重抗病毒之策略。為了產生對 ZYMV, PRSV-W 以及 CMV 的抗性,我們的構築取自三種病毒之鞘蛋白基因(coat protein, CP) 序列片段與西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV) 的基因默化誘發序列經設計接合成的非轉譯性基因融合載體(chimeric vector)。此基因融合載體再藉由農桿菌 (Agrobacterium) 的轉型作用產生瓜類轉基因植株。轉基因甜瓜株系進行多重病毒抗性評估發現對於ZYMV以及PRSV-W具有高度抗性;相對於ZYMV和PRSV-W,CMV具較低程度之抗性。根據結果顯示此多重基因構築可以提供病毒多重抗性,將可成功的應用在重要的經濟作物上。我們進一步的比較不同轉基因構築體之結構對於提供多重抗病毒效率之影響. 本研究中,我們分別轉殖三種不同結構之構築到甜瓜中,包括 short sense, short inverted repeat 以及 long sense 之ZYMV, PRSV-W及CMV病毒片段複合之構築。三種不同結構之構築載體誘導不同程度之基因沉寂效率,short inverted repeat 和long sense之構築能夠引發較強的基因沉寂效率,故我們可藉此設計適合的構築去提高基因沉寂之效率。轉殖此三種構築之轉基因甜瓜抗病株系具有較低的RNA累積並可偵測到short-interfering RNAs (siRNA)的產生,此結果更進一步的印證轉基因抗病機制來自於後轉錄基因沉寂之理論。我們的研究預期將來可應用在葫蘆科以外的作物並幫助培育具有廣泛病毒抗性或抗其他病原菌之重要經濟作物
Since most crops in the field are prone to attack by several viruses, development of technologies which could confer resistance to multiple viruses can help reduce agricultural losses to a great extent. Cucurbitaceae is a commercially important family of crop plants around the world that are subject to severe economic losses due to an array of distinct viral infections. Three major viruses that affect cucurbits production are Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus type watermelon (PRSV-W), and Cucumber mosaic virus (CMV). Mixed virus infections are quite often detected in cucurbits which further enhance the severity of crop damage. Therefore, it is highly desirable to develop virus resistant cultivars, especially which can combat multiple virus infection, making use of the advanced genetic engineering approaches. The main objective of the work presented in this thesis was directed toward the generation of broad-spectrum resistance against three highly destructive viruses i.e. ZYMV, PRSV-W, and CMV, members of two distinct viral groups, in melon (Cucumis melo L.), based on post-transcriptional gene silencing mechanism. This dissertation is divided into four chapters. Chapter 1 describes all revelent references for this study. In chapter 2 we demonstrate that fusing transgene segments of different viruses in a chimeric form along a gene silencing inducing sequence, designated as silencer DNA, represent an efficient strategy for generating multiple virus resistance. To generate triple virus resistance, a single chimeric vector consisting of partial non-translatable coat protein (CP) gene sequences of ZYMV, PRSV-W, and CMV coalesced along the silencing inducing sequence of Watermelon silver mottle virus (WSMoV) was designed. The chimeric vector was used for the generation of transgenic melon using Agrobacterium-mediated transformation. Transgenic melon lines assessed for multivirus resistance exhibited higher degrees of resistance against the two potyviruses (ZYMV and PRSV-W) and a lower, but significant, protection to CMV. The results indicated that pyramiding gene segments from different viruses in a chimeric form can be successfully applied for developing simultaneous resistance against several viruses in economically important cultivars. In chapter 3 we further extended our study to compare the influence of transgene architecture on the relative accessibility of chimeric gene constructs for generating multiple resistance against targeted viruses. Three different chimeric constructs expressing the CP gene segments of ZYMV, PRSV-W, and CMV fused together in short sense, short inverted repeat and long sense form were developed. Transgenic melon lines, generated for each construct, showed a considerable difference in silencing efficiencies against homologous viruses. The results indicated that silencing was stronger when initiated by chimeric transgene arranged in short inverted repeat, or longer sense, form then by shorter sense transgene. These results demonstrate that efficiencies of chimeric transgenes against multiple targets can be elevated by arranging them in favorable architectural forms. The silencing induced by different chimeric constructs used in this study (Chapter 2, 3) was associated with reduced transgene transcript accumulation, indicating the role of RNA mediated resistance. Detection of short-interfering RNAs (siRNAs), corresponding to different subunits of the chimeric transgene exclusively in virus resistant plants, further confirmed that resistance was indeed achieved at the post-transcriptional level. Chapter 4 describes the conclusions and future prospects of this study. Overall, we assume that the outcome of this research work can be successfully applied to other plant species, apart from cucurbits, that could be helpful to generate resistant crops not only against viruses, but also to other harmful pathogens.
其他識別: U0005-2508200815365700
Appears in Collections:植物病理學系



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