Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/37274
標題: 建立病毒誘導基因沉寂系統進行圓葉菸草多元胺生合成相關基因之分析
Development of virus-induced gene silencing system to study polyamine synthesis-related genes in Nicotiana benthamiana
作者: 林堂煌
Lim, Tang-Huang
關鍵字: 病毒誘導基因沉寂
virus-induced gene silencing
多元胺
鳥胺酸脫羧酶
甲基茉莉酸
脯胺酸
polyamine
ornithine decarboxylase
methyl jasmonate
proline
出版社: 農藝學系所
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摘要: 本實驗室目前具有由T7啓動子調控菸草嵌纹病毒 (Tobacco mosaic virus, TMV) 之感染性選殖株 (infectious clone),由於該系統的缺點為操作過程繁瑣、藥品昂貴以及RNA容易被降解而可能影響試驗的穩定性,本研究將TMV全長度基因體構築於Ti-質體 (tumor inducing plasmid, Ti-plasmid),成功建立生體内 (in vivo) 感染性選殖株的系統。多元胺 (Polyamines, PAs) 是一類具有生物活性的低分子脂肪族陽離子,存在於所有生物體中。鳥胺酸脫羧酶 (ornithine decarboxylase, ODC) 與精胺酸脫羧酶 (arginine decarboxylase, ADC) 是已知合成多元胺的兩種酵素。從即時反轉錄聚合酶鏈鎖反應 (real-time Reverse Transcription-Polymerase Chain Reaction, real-time RT-PCR) 的結果發現施用甲基茉莉酸 (methyl jasmonate, MeJ) 會大量誘導ODC基因的表現,但其詳細調控機制目前尚未確認。本論文利用所建立的生體内TMV感染性選殖株系統作爲病毒誘導基因沉寂 (virus-induced gene silencing, VIGS) 的工具,分別降低圓葉菸草 (Nicotiana benthamiana L.) 體内ODC及ADC的表現,並進一步利用MeJ誘導植物累積polyamine,從分析結果推測MeJ主要透過ODC的途徑生成腐胺 (putrescine)。由於ODC的反應物為鳥胺酸,鳥胺酸亦可用於脯胺酸 (proline) 的合成,將TMV-ODC分別處理29%聚乙二醇 (polyethylene glycol, PEG) 及0.1 mM離層酸 (abscisic acid, ABA),結果顯示在29% PEG處理下,ODC的表現受到抑制有助於提供更多的鳥胺酸供脯胺酸合成,但離層酸的處理則不會,因而確認OAT (ornithine-δ-aminotransferase) 僅參與滲透逆境誘導脯胺酸累積的角色。
The T7 promoter-driven Tobacco mosaic virus (TMV) infectious clone was currently practiced in our lab. The disadvantages of this system are the cumbersome procedure, expensive chemicals and the easily degraded RNA, which affect the stability of the experiments. In this thesis, we constructed the full-length cDNAs of TMV into Ti-plasmid and successfully established an in vivo system of TMV infectious clone. Polyamines (PAs) are low molecular weight, aliphatic polycations found in the cells of all living organisms. Polyamines could be synthesized with ornithine decarboxylase (ODC) from ornithine and with arginine decarboxylase (ADC) from arginine in plants. The result of real-time RT-PCR implicated that methyl jasmonate could induce the expression of ODC gene, but its mechanism is still unclear. The aim of this thesis is to use TMV infectious clone as a virus-induced gene silencing tool to lower the activities of ODC and ADC in Nicotiana benthamiana L. and further use methyl jasmonate to induce the accumulation of PAs. Results showed that ODC is a major enzyme for putrescine accumulation when methyl jasmonate is applied. The fact that ornithine can be used for the synthesis of PAs via ODC and for the synthesis of proline via ornithine-δ-aminotransferase (OAT) provides a system for the investigation of the crosstalk between ODC and OAT. Leaf discs containing TMV-ODC were treated with 29% PEG or 0.1 mM abscisic acid (ABA). Results showed that the ODC activity was inhibited when treated with 29% PEG resulting in the accumulation of higher proline level via OAT, but no significant difference was observed when treated with 0.1 mM ABA. Thus, the role of OAT participating only in the osmotic stress-induced proline accumulation was confirmed.
URI: http://hdl.handle.net/11455/37274
其他識別: U0005-2208201316093900
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