Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31535
標題: 利用針對病毒高序列保留區之人工微小RNAs產生廣泛抗馬鈴薯Y屬 病毒之轉基因煙草
Transgenic tobacco with resistance to potyvirus generated by artificial miRNAs targeting at highly conserved regions of potyviral genomes
作者: 帝瑪
Dmytro, Ustianenko
關鍵字: artificial miRNAs
人工微小RNA
出版社: 植物病理學系所
摘要: 馬鈴薯Y 屬病毒是植物病毒屬中最大也是經濟上最重要的屬,本研究之目的是利用 人工微RNA (artificial miRNA, amiRNA) 的策略產生具有高度抗馬鈴薯Y 屬中不同病毒的轉 基因植物。為了產生具廣泛抗性的轉基因,比對了馬鈴薯Y 屬中16 條不同病毒全基因體 並找出高度保留區。含有273 個鹼基對的阿拉伯芥的微RNA159a (miRNA159a)被選為設計 的骨架,以用於構築人工微RNA,利用寡核苷酸直接突變 (oligonucleotide-directed mutagenesis) 的方式,將產生微RNA 的21 個核苷酸區域取代成可配對至CI、NIb 或CP 基 因高度保留區的21 核苷酸序列。六個帶有單套、一個帶有雙套、及一個帶有參套的人工 微RNA 前趨物被構築。這些突變改造完成的人工微RNA 進一步選殖至貳位元載體 (binary vector) pBA-DC-HA 或pB2T-DC-HA,為了測試這些人工微RNA 能否表現,抽取經過農桿 浸染 (agro-infiltrated) 的全RNA (total RNA),並用專一性探針以北方墨點法測定相對應的人 工微RNA 的表現,我們的結果指出所有的人工微RNA 構築,皆能有效的表現人工改造的 微RNA 於農桿菌浸染的葉片中。所有的人工微RNA 構築便以農桿菌媒介的方式將這些人 工微RNA 轉殖至菸草中。所產生的大量的植株先以蕪菁嵌紋病毒 (Turnip mosaic virus, TuMV) 在溫室的條件下分析其抗性,有些植株具有延遲發病的效果,其中一個帶有三套 人工微RNA 的轉基因展現了高度的抗病性,這證明了人工微RNA 針對病毒高度保留區是 一個很好的策略以用於生產抗病的轉基因植物,其實際廣泛性的抗病效益尚待進一步測 試。
The genus Potyvirus is the largest and economically most important plant virus group. The objective of this study was directed to generate transgenic plants with a high level of resistance against different potyviruses using the artificial micro RNA (amiRNA) approach. In order to create a broad-spectrum resistance to different viruses, highly conserved regions among 16 potyviral genomes were identified by sequence alignment. The 273-nt pre-miR159a from Arabidopsis was chosen as the backbone for the construction and expression of amiRNAs. Using oligonucleotide-directed mutagenesis, a 21 nucleotide region of miR159 was replaced by a synthetic 21-nucleotide sequence targeting at the highly conserved regions of potyviral CI, NIb, or CP genes. Six single, one double and one triple precursor amiRNAs were created. The constructed sequences were then moved to the binary vector pBA-DC-HA or pB2T-DC-HA. To check the expression of designed amiRNAs, the total RNA was isolated from agro-infiltrated leaves and detected by northern blotting with a specific probe against the corresponding amiRNA sequence (21nt). Our results revealed that all constructed amiRNAs in pre-amiRNA constructs were efficiently expressed in agroinfiltrated leaves. Agrobacterium-mediated transformation method was used to transform plants of Nicotiana benthamiana. Transgenic lines were obtained and their resistance against potyvirus was evaluated by mechanical challenge with Turnip mosaic virus under greenhouse conditions. Some transgenic lines showed symptom delay in comparison to non-transgenic plants. One transgenic line expressing the constructed three amiRNAs exhibited a high level of resistance, indicating that the amiRNA approach targeting at highly conserved regions of potyviral genomes is a good strategy for generating transgenic resistance against potyvirus. Whether the transgenic lines provide broad-spectrum resistance against different potyviruses remains to be further tested.
URI: http://hdl.handle.net/11455/31535
Appears in Collections:植物病理學系

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