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研究生:吳偉誌
研究生(外文):Wei-Zhi Wu
論文名稱:利用VIGS靜默多元胺三胺合成酶基因探討多元胺與一氧化氮對圓葉煙草(Nicotiana benthamiana)根部生長的影響
論文名稱(外文):Investigating the roles of polyamine-spermidine biosynthetic enzyme and nitric oxide in root growth of Nicotiana benthamiana by virus-induced gene silencing
指導教授:林忠毅林忠毅引用關係
指導教授(外文):John-Yee Lin
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:98
中文關鍵詞:多元胺一氧化氮病毒誘導基因靜默圓葉煙草
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多元胺(Polyamines)對植物的生理與發育過程扮演重要的角色,另外一個參與植物逆境相關性物質則是一氧化氮(Nitric oxide),兩者訊息分子之間於路徑上具有一個潛在的調節特定相關性。目前一氧化氮與多元胺兩者主要為調解植物生理的養分平衡及活性氧(reactive oxygen species)的產生,並且彼此為互相誘導累積及時間序列上具有特定之交感作用,其之間作用機制仍需研究。Virus-induced gene silencing (VIGS)技術提供探討植物的基因功能,本研究目的主要探討VIGS靜默多元胺三胺合成酶基因靜默後,對圓葉煙草根部生長形態的影響,以及探討多元胺與一氧化氮扮演的角色。
研究結果顯示VIGS技術經由葉片感染,擴散到根部具有靜默的效應。經由reverse transcription-PCR與real time PCR證實三胺合成酶基因靜默後,其基因表現量確實下降。此外,多元胺三胺合成酶(SPDS)靜默後對圓葉煙草根部的效應,以HPLC分析根部內源性多元胺含量,發現三胺含量下降伴隨著二胺含量上升,且利用螢光比色儀測定一氧化氮含量有上升的趨勢。三胺合成酶基因靜默後會影響圓葉煙草根部的生長形態,即抑制根部鮮重與總長度。最後本研究確認該鹽分逆境下之多元胺合成酶基因靜默的可行性,結果顯示鹽分下,VIGS技術研究可靜默三胺合成酶基因表現。
綜合上述結果,本實驗研究發現多元胺三胺合成酶靜默後,會影響圓葉煙草多元胺與一氧化氮生理之生化合成路徑及根部生長形態的改變,並確認鹽分逆境下,VIGS技術的可行性。
Polyamines and the biosynthesis of the corresponding enzymes have been implicated in environmental stresses and plant growth. Another NO (Nitric oxide) is a highly diffusible gaseous free radical involved in plant stress-related substance. However, the roles of polyamine and nitric oxide in root growth have not been fully elucidated. In this study, Nicotiana benthamiana was used as a model plants to investigate the roles of polyamine in root growth. Genes encoding polyamine biosynthesis enzymes were knocking down by using the virus-induced gene silencing (VIGS) strategies. Analysis of root growth was carried out in the spermidine synthase (SPDS)-silenced plants. In NbSPDS-silenced plants, it was observed that the number of lateral roots was decreased as compared to vector-control plants. The effects of VIGS on gene expression level were validated by using reverse transcription-PCR. The expression of genes encoding Tobacco rattle virus (TRV) coat protein was detected in the VIGS-treated root tissues. When compared with the vector-control lines, the expression of NbSPDS gene was significantly decreased. The NO level was higher in the NbSPDS-silenced plants than that of vector-control and wild-type plants. High performance liquid chromatography analysis of polyamine content showed that the content of spermidine was decreased in the NbSPDS-silenced plants as compared to the vector-control plants. Moreover, the VIGS technology was apply for abiotic stress. In summary, this study suggests that spermidine may play important roles in root growth.
目錄
中文摘要 2
英文摘要Abstract 4
致謝 7
縮寫表 8
1.前言 9
1-1.多元胺對植物生長發育及生理生化的影響 9
1-2.一氧化氮對植物生長發育及生理生化特性 14
1-3.多元胺與一氧化氮之間的關聯性 21
1-4.利用病毒誘導基因靜默探討多元胺靜默對植物生長發育的作用 27
1-5.研究目的 33
2. 材料方法 34
2-1.植物的生長條件與處理 (growth and treat conditions) 34
2-2.VIGS(virus-induced gene silencing)前置作業 35
2-3.病毒誘導基因靜默的進行 36
2-4.圓葉煙草形態觀察、鮮重總長度分析及轉移感染觀察 37
2-5.靜默後基因表現量分析 38
2-6.萃取多元胺及分析其含量 41
2-7.利用螢光染劑與一氧化氮結合分析其含量 42
2-8.統計分析 43
3. 結果 44
3-1.VIGS病毒可否感染圓葉煙草根部的檢測 44
3-2.多元胺三胺合成酶靜默後對圓葉煙草根部生長形態的影響 49
3-3.利用VIGS技術靜默多元胺三胺合成酶於圓葉煙草根部的效應 51
3-4.評估鹽逆境下圓葉煙草根部之多元胺合成酶基因靜默的效應 59
4. 討論 65
4-1. VIGS病毒可否感染圓葉煙草根部的檢測 65
4-2.多元胺三胺合成酶靜默後對圓葉煙草根部生長形態的影響 68
4-3.利用VIGS技術靜默多元胺之三胺合成酶於圓葉煙草根部的效應 71
4-4.評估鹽逆境下圓葉煙草根部之多元胺合成酶基因靜默的效應 79
5. 結論 83
6. 參考文獻 84
7. 附錄 96


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