Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31318
標題: 利用基因沉寂機制產生具廣泛抗兩種馬鈴薯Y屬病毒轉基因木瓜及利用人工微小核醣核酸攻擊番茄斑萎屬病毒複製酶基因以產生抗病能力
Improvement of transgenic papaya with broad-spectrum resistance to two potyviruses by siRNA approach and expression of artificial microRNAs targeting tospoviral replicase gene to generate virus resistance
作者: 龔怡蓉
Kung, Yi-Jung
關鍵字: 木瓜輪點病毒
Papaya ringspot virus
木瓜畸葉嵌紋病毒
轉基因木瓜
後轉錄基因沉寂作用
不定根體胚
人工微小核醣核酸
Papaya leaf-distortion mosaic virus
transgenic papaya
post-transcriptional gene silencing
somatic embryos of adventitious roots
artificial microRNAs
出版社: 植物病理學系所
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摘要: 木瓜為熱帶及亞熱帶地區重要的水果之ㄧ,其產量受到木瓜輪點病毒 (Papaya ringspot virus, PRSV ) 感染而驟減。此病毒在無化學防治方法及無天然抗病品種可利用下,本研究室以基因工程技術成功發展本土PRSV 嵌紋型YK 病毒株之鞘蛋白 (coat protein, CP) 轉基因木瓜,結果顯示對本土及各區域之PRSV具有非常良好的抗性。此轉基因木瓜為台灣第一個通過農委會 (Council of Agriculture, COA) 田間試驗之轉基因作物。於田間試驗期間,發現另一病毒會感染此YK-CP轉基因木瓜,此病毒與PRSV並無血清關係,經鑑定後發現為不同於PRSV的另一馬鈴薯Y屬病毒 (Potyvirus) ─ 木瓜畸葉嵌紋病毒 (Papaya leaf-distortion mosaic virus, PLDMV)。除了PLDMV,田間試驗期間亦發現另一PRSV 強系病毒會擊垮YK-CP轉基因作物,命名為 PRSV 5-19。因此,在本論文中,我們主要目的為發展雙抗PRSV及PLDMV和超抗PRSV 5-19之轉基因木瓜,解決木瓜病毒病害之問題,配合新發展的農桿菌轉殖法,以兩性株木瓜不定根體胚為轉殖材料可縮短固定性狀之育種時間。最近轉基因抗病毒策略有重大突破,利用人工合成之微小核醣核酸 (artificial microRNA, amiRNA) 可成功應用來抵抗正股核酸病毒,因此,本論文也利用此策略設計amiRNA 攻擊番茄斑萎病毒屬之負股核酸的複製酶 (replicase) 基因達到抗病毒能力。 本論文第一章為木瓜生物技術及轉基因抗病機制之文獻回顧。第二章為「雙重抗木瓜輪點病毒及木瓜畸葉嵌紋病毒轉基因木瓜之育成」,為有效防治PRSV及PLDMV,以此兩病毒部份鞘蛋白基因以非轉譯性方式構築,以先前未知性狀之木瓜未成熟胚為材料利用農桿菌進行轉殖,我們成功得到數個轉基因木瓜對此兩病毒皆顯示良好抗性,其中三個品系對夏威夷、泰國及墨西哥PRSV分離株具有廣泛性抗性,此抗性是藉由後轉錄基因沉寂作用 (post-transcriptional gene silencing, PTGS) 所提供。然而,此轉基因木瓜種植六個月之後發現皆為雌株 (female),非消費者喜愛之兩性株 (hermaproditic),因此需花費許多育種時間固定性狀。 本論文第三章為「利用木瓜不定根體胚育成具抗病毒性狀之兩性株轉基因木瓜」,為縮短育成具良好園藝性狀的抗病兩性株木瓜之時間,本研究選定優良性狀台農二號兩性株木瓜,經微體繁殖及不定根生長,找出由不定根誘導出體胚之最適條件,以此兩性株體胚取代先前未知性別之未成熟胚為轉殖材料,利用農桿菌進行轉殖,我們可以在七個月內得到具兩性性狀之新雙抗台農二號木瓜,此木瓜可直接以微體繁殖方法將其商品化。以此方法亦成功應用於台農二號親本日陞種及泰國種兩性株木瓜,發展抗PLDMV之親本轉基因木瓜,此可作為增強抗病之親本,此新轉殖方法大大縮短育種時間並有效應用於其他品種木瓜。 第四章為「非轉譯基因沉寂抑制子轉基因木瓜解決非依賴性同源性抗性之問題」,最近的實驗證明PRSV超強病毒株5-19擊垮YK-CP轉基因木瓜之抗性為其超強基因沉寂抑制子 (gene silencing suppressor) 所致,此超強病毒亦會擊垮本研究第二章發展之雙抗轉基因木瓜,因此為發展轉基因抗此超強病毒株,本研究以非轉譯性HC-Pro基因配合第三章之不定根體胚轉殖法,以農桿菌為媒介轉殖至木瓜中,藉由植物基因沉寂機制直接將病毒抑制子基因瓦解而達抗超強病毒能力,本研究得到之轉基因木瓜對PRSV超強病毒株5-19及本土嵌紋型病毒株YK具有良好之抗性,亦對其他地區PRSV病毒株系具有非常良好抗性,可解決依賴性同源性抗性被強系病毒擊垮之問題。此超抗木瓜將來可與本研究中發展之雙抗木瓜雜交發展出具全球應用價值之雜交子代。 第五章為「利用人工微小RNA攻擊負股RNA病毒複製酶基因之高保留區達到抗病能力」,為發展番茄斑萎病毒屬病毒抗病植物,本研究設計多個amiRNA 互補西瓜銀斑病毒 (Watermelon silver mottled virus, WSMoV) 之負股 L RNA 複製酶基因高保留區域。於單一amiRNA 及三個組合的amiRNAs轉基因植物中我們成功的得到對WSMoV具抗性之轉基因菸草,其中三個amiRNAs轉基因菸草對WSMoV具高度抗性,此為第一篇利用amiRNA策略攻擊複製酶基因得到對負股病毒具抗性之報導,可藉由與基因沉寂抑制子NSs基因高保留區域amiRNA轉基因菸草雜交,得到廣泛抗番茄斑萎病毒屬之轉基因植物。
Papaya is one of the important tropic and subtropic fruits. The production of the fruit is seriously limited by infestation of Papaya ringspot virus (PRSV) worldwide. Since chemical control measures are not feasible and no natural resistance sources are available for conventional breeding, our laboratory has successfully developed a plant genetic engineering approach to generate transgenic papaya lines that carry the coat protein (CP) gene of Taiwan mosaic strain PRSV YK and confer high levels of resistance to PRSV infection. These transgenic papaya lines were approved by the Council of Agriculture (COA) as the first case for field tests of transgenic crop in Taiwan. During the field tests of PRSV YK CP transgenic papaya lines, we discovered an unrelated potyvirus, Papaya leaf-distortion mosaic virus (PLDMV), which was able to break down the resistance provided by the PRSV YK CP transgene. Moreover, we also found a super strain of PRSV, isolate 5-19, was able to overcome the CP- transgenic resistance. In this dissertation, for control these viruses and shortening breeding time, we develop transgenic papaya lines with double-virus resistance to PRSV and PLDMV and super resistance to PRSV 5-19, via transformation of somatic embryos derived from adventitious roots of in vitro shoots. Recently, a novel strategy for plant virus resistance using artificial microRNA (amiRNA) was developed. Here, we also developed several amiRNAs targeting the conserved motifs of the replicase gene (negative-sense ssRNA) of a tospovirus to confer virus resistance. In this dissertation, the Chapter 1 “Literature review” describes all relevant references for papaya biotechnology and resistance mechanism. Also, the background and rationale of each study, and the approaches used to solve the problems were described. In order to overcome the potential threat of PLDMV, the Chapter 2 described the development of papaya transgenic lines that carry a chimeric construct with parts of CP coding sequences of both PRSV YK and PLDMV, conferring double resistance to both PRSV YK and PLDMV is described. These double-resistance lines were evaluated under greenhouse conditions. Several lines with complete resistance to PRSV and PLDMV were obtained. Furthermore, three of nine resistant lines showed high levels of broad-spectrum resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. However, this attempt ended up in the production of resistant lines (R0) displaying female sex, on attaining flowering stage after being reared for six months under greenhouse conditions. In Chapter 3, in order to shorten the time-consuming breeding program for fixing the transgenic resistance and the hermaphroditic sex, a novel transformation method was attempted. The somatic embryos derived from the adventitious roots of in vitro shoots of selected hermaphroditic Tainung No. 2 plants, the most popular commercial hybrid cultivar in Taiwan, were used as explants for transformation. Using our protocol, a commercially valuable papaya variety with hermaphrodite sex and double-virus resistance to PRSV and PLDMV can be regenerated within seven months. For hybrid-breeding purpose, this method was also successfully applied to deliver an untranslatable PLDMV CP construct to Tainung No. 2 parental cultivars Thailand and Sunrise, both with hermaphrodite sex, to generate resistance to PLDMV. Our protocol can avoid time-consuming and labor-intensive breeding programs for producing elite cultivars of transgenic papaya hybrid with resistance to both PRSV and PLDMV. Our recent evidence showed that PRSV 5-19 contains a strong gene silencing suppressor HC-Pro that can suppress transgenic resistance mediated-through gene-silencing in a sequence homology independent manner. In Chapter 4, we intended to develop new transgenic papaya lines resistant to the super strain PRSV 5-19 to ease its potential threat on the breakdown of the single-virus or double-virus transgenic resistance. For targeting HC-Pro gene of PRSV 5-19 through RNA-mediated gene silencing, new transgenic papaya lines carrying the untranslatable HC-Pro coding sequences were developed by Agrobacterium-mediated transformation of somatic embryos derived from adventitious roots of in vitro shoots of selected papaya individuals with hermaphrodite sex and desirable properties. These new papaya transgenic lines confer complete resistance to PRSV 5-19 and other PRSV strains from Taiwan or other geographic origins. After crossing with double-virus resistant papaya, the hybrid progenies are expected to have a great potential for global application for control of PRSV and PLDMV. In Chapter 5, for control of negative sense RNA virus, transgenic plants expressing amiRNAs targeting the conserved regions of tospoviral replicase genes were generated. The results indicated that two valid amiRNAs, out of the six designed amiRNAs were found to be effective against Watermelon silver mottle virus (WSMoV), when expressed either as single or triple amiRNAs in transgenic N. benthamiana lines. Our triple amiRNA constructs provided complete resistance to homologous tospovirus. This is the first report for the management of negative-sense plant RNA viruses using amiRNA strategy which targets the replicase gene highly conserved motif of tospoviruses. In future, we will combine the triple amiRNAs targeting the L gene with amiRNA targeting the common epitope of the NSs gene silencing suppressor, to offer broad- spectrum resistance against different serogroups of tospoviruses. Also, this amiRNA approach can be extended to real crops against different plant viruses and thus could play an important role in sustainable agriculture.
URI: http://hdl.handle.net/11455/31318
其他識別: U0005-1908200910325600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1908200910325600
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