臺灣博碩士論文加值系統

本研究中選用茄科植物圓葉煙草 (Nicotiana benthamiana) 為材料，利用VIGS (virus induced gene silencing) 技術靜默多元胺合成酶基因，觀察不同多元胺合成酶基因被靜默後，對植物生長發育的影響。此外實驗中也使用綠螢光蛋白標記三胺合成酶基因，觀察目標基因作用位置並推導其機制。
在煙草中目標基因靜默後，本研究藉由RT-PCR觀察各煙草多元胺合成酶基因的表現量。從實驗結果得知，將二胺合成酶分別為ADC (arginine decarboxylase)、ODC (ornithine decarboxylase) 及三胺合成酶SPDS (spermidine synthase)靜默後，均獲得該基因之表現量確實明顯下降。SPDS基因靜默圓葉煙草，植物有矮化的症狀，此外明顯的有花期延後的現象，花柱的發育亦受到影響，成熟果莢數目明顯減少，ADC及ODC基因的靜默圓葉煙草則無明顯差異。藉由RT-PCR分析多元胺基因在圓葉煙草不同組織之表現量，結果發現NbADC基因在圓葉煙草年輕葉、花、根、頂芽的組織有較高之表現量，而NbODC、NbSPDS兩個基因在花、頂芽組織有較高的表現量，以HPLC分析靜默後多元胺表現量的結果也與基因的表現量的結果一致。實驗中並使用綠螢光蛋白標記三胺合成酶基因，認為三胺合成酶可能分佈於細胞膜上。經由上述研究結果，發現Spd參與繁殖器官生長發育之調控較為顯著。

Polyamines are implicated in a wide array of fundamental processes in plants such as adaptation and tolerance to environmental stresses. However, their roles in plant growth and developmental programs have not been fully elucidated. In this study, Nicotiana benthamiana was used as a model plants to investigate the roles of polyamine biosynthesis in developmental programs. Genes encoding polyamine biosynthesis enzymes were knocking down by using the VIGS (virus-induced gene silencing) strategies. Analysis of plant growth and development programs was carried out in these gene-silencing plants. Subcellular localization of the polyamine biosynthesis enzyme labeled with green fluorescent protein was performed to observe the localization and deduce the mechanism of action.
The effects of VIGS on gene expression level were validated by RT-PCR. When compared with the vector-control lines, the expression of genes encoding diamine synthase ADC (arginine decarboxylase), ODC (ornithine decarboxylase) and synthase triamine SPDS (spermidine) was significantly decreased. In NbSPDS-silenced plants, the growth and developmental programs were impaired such as dwarfing, delayed flowering, abnormal style development and reduced the number of fruit capsules. The NbADC- and NbODC-silenced plants exhibited normal morphology. By RT-PCR analysis of polyamine gene expression in different tissues, it was found that NbADC gene was expressed in young leaves, flowers, roots and shoot apex. The expression of NbODC and NbSPDS genes were relatively higher in flower and shoot apex. HPLC analysis of polyamine content showed that the content of spermidine was decreased in the NbSPDS-silenced plants when compared with the vector-control plants. In summary, this study suggests that spermidine plays an important role in the regulation of growth and developmental programs in plants.

目錄
中文摘要 2
英文摘要Abstract 4
致謝 6
縮寫表 9
1. 前言 10
1-1. 植物中多元胺之代謝生理 10
1-2. 多元胺對植物生長發育的影響 13
1-3. 逆境下多元胺對生理表現的影響 18
1-4. 利用病毒誘導基因靜默進行多元胺靜默對植物生長發育的影響 20
1-5. 研究目的 24
2. 材料方法 26
2-1. 植物的生長條件與處理 (growth and treat conditions) 26
2-2. VIGS (virus-induced gene silencing) 前置作業 27
2-3. 病毒誘導基因靜默的進行 37
2-4. 三胺合成酶融合綠螢光蛋白 (SPDS-GFP) 構築與細胞表現位置觀察 38
2-5. 萃取多元胺及分析其含量 40
3. 結果 42
3-1. 利用VIGS技術對多元胺合成酶基因靜默的效應 42
3-2. 多元胺合成酶基因靜默後對植物生長發育的影響 49
3-3. 多元胺合成酶基因表現的組織特異性 54
4. 討論 59
4-1. 利用VIGS技術對多元胺合成酶基因靜默的效應 59
4-2. 多元胺合成酶基因靜默後對植物生長發育的影響 64
4-3. 多元胺合成酶基因表現的組織特異性 67
5. 結論 70
6. 參考文獻 72
7. 附錄 78
7-1. 多元胺生合成路徑 (Takahashi T. and Kakehi J., 2010) 78

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