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研究生:帝瑪
研究生(外文):Ustianenko Dmytro
論文名稱: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
論文名稱(外文):Transgenic tobacco with resistance to potyvirusgenerated by artificial miRNAs targeting at highlyconserved regions of potyviral genomes
指導教授:葉錫東
指導教授(外文):Shyi-Dong Yeh
學位類別:博士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
畢業學年度:96
語文別:英文
論文頁數:44
中文關鍵詞:人工微小RNA
外文關鍵詞:artificial miRNAs
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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.
Abstract……………..………………………………………….………………… 1
中文摘要…………………………………………………………………….……. 2
Introduction…………………………………………………………………….... 3
Materials and methods……………………………………………………….… 9
Sequence alignment and miRNA design……………………….…………… 9
Construction of single artificial pre-miRNA.………………..………………. 9
Construction of double pre-amiRNA……….……………..……………….. 10
Construction of triple pre-amiRNA……….……………………………..…… 10
Transient expression by agro-infiltration of N. benthamiana………………... 11
Plant Transformation……………………………………………………........ 11
Northern hybridization……………………………………………………… 12
Resistance evaluation………………………………...………………….….. 12
Detection of the TuMV in infected transgenic lines by indirect enzyme-linked
immunosorbent assay (ELISA)…………………………………….…........... 13
Results………………………………………………………….………………… 14
Construction of single artificial miRNA…..……………………….………… 14
Construction of double amiRNA…………….…………………….…………. 14
Construction of triple amiRNA…………………………………….……….... 14
Transient expression of amiRNA in leaves of Nicotiana
benthamiana ..................................................................................................... 15
Establishment of transgenic lines…………………………….……………… 15
The expression levels of artificial miRNA in transgenic tobacco
plants ………………………….…………………………………………..... 16
Resistance evaluation in transgenic tobacco plants…………………….…… 16
III
Discussion…………………………………………………..………………... 18
References……………………………………………………………………. 21
Figures and tables..…………………………………………………………… 29
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