臺灣博碩士論文加值系統

本研究的目的在探討不同種類品系的植物在不同的反應溫度、胞器、生長天數、根的部位、季節變遷、抑制劑(Acriflavin )、pH 值、CuCl2 等的變化對cytokinin oxidase活性的影響。因此實驗中改變上述的變因以分析植物體內cytokinin oxidase的活性。結果如下：
1. 不同種類的植物其cytokinin oxidase的活性對於不同反應溫度將呈現出截然不同的走勢。如：胡瓜的根在37oC、43oC 活性為零，直到45 oC則活性大增。甜豌豆的根在37oC活性很低，到43oC則活性大增，再提高到45 oC則活性降為0。玉米在這三個溫度活性略有起伏，但差異並不大。稻苗除了台梗14號在37oC有最佳的活性外，其他多數在43oC有最佳的活性，至於溫度升到45oC則活性又驟降，(除了台梗5號及台梗8號以外)。
2. 不同種類品系的植物其cytokinin oxidase活性將分佈在不同的胞器內，並且差異極大。如：台梗3號只在細胞質有活性；台梗9號在細胞質、粒線體、細胞壁上有活性，其中以粒線體的活性為最高。玉米根在細胞質、粒線體、細胞壁上皆有活性。胡瓜的根則在細胞質、細胞壁有高度的活性。
3. 植物的幼苗期cytokinin oxidase的活性隨著生長天數呈現逐漸增加的情形，直到成熟期達到最頂峰後又驟降。
4. cytokinin 主要是在根尖合成的，cytokinin oxidase的活性也隨之在根尖的部位遠比在根的成熟部活性來得高出許多。
5. 季節對植物cytokinin oxidase的活性影響則因不同植物而異，有些植物如：水稻Tai-keng 9在夏季cytokinin oxidase的活性較高，有的植物如：胡瓜Pretty swallow則在冬季cytokinin oxidase的活性則較高。
6. 抑制劑(Acriflavin)在玉米根的cytosol部份有極明顯的抑制作用，但在細胞壁的部分沒有抑制效果。
7. 水稻在 pH值為8時，cytokinin oxidase的活性最佳。
8. 不同CuCl2 濃度皆有促進cytokinin oxidase活性的效果，其中以濃度為5 mM時有最佳的促進效果。

This thesis mainly studied cytokinin oxidase activity in several plant species. Several factors which affect this enzyme activity were involved in this research: species, assay incubation temperatures, subcellular locations, tissue age, root tissue portion, season, inhibitor of flavoprotein (Acriflavin), pH values and CuCl2 concentrations of the reaction medium. Results are outlined as the following:
1. The response of cytokinin oxidase activity to assay incubation temperatures is different among plant species and varieties. Cytokinin oxidase of cucumber seedling roots show zero activity at assay temperatures 37oC and 43 oC, but increasing greatly at 45oC. Cytokinin oxidase of pea seedling roots has high activity at 43oC, but decreases to zero at 45 oC. Cytokinin oxidase in corn seedling roots of three varieties show low activity at 37-43 oC. Rice seedling shoots of 10 rice varieties exhibit cytokinin oxidase function at 37-43 oC, but decrease activity at 45 oC, except Tai-keng 5 and Tai-keng 8.
2. Cytokinin oxidase locations in subcellular fractions are also variable in different plant species and varieties. Cytokinin oxidase of Tai-keng 3 rice seedling shoots only shows its activity in cytosol fraction, while cytokinin oxidase of Tai-keng 9 displays activity in fractions of mitochondria, cell wall and cytosol. In corn seedling roots, cytokinin oxidase demonstrates activity in 3 fractions, including mitochondria, cell wall and cytosol in two varieties, (Morrison and Bright jean), while cytokinin oxidase from Honey jean 2 only shows activity in cell wall and cytosol fractions. Cytokinin oxidase of cucumber seedling roots manifests activity in cell wall and cytosol, but cytokinin oxidase of seedling cotyledons only shows activity in cell wall. Roots of pea seedlings have cytokinin oxidase in three fractions, cell wall, cytosol and mitochondria.
3. High activity of cytokinin oxidase is correlated with active growth stage during seedling growth.
4. High cytokinin oxidase activity is located in young tissue part of corn seedling roots.
5. The activity of cytokinin oxidase in plant seedlings is in connection with seasonal changes: In Tai-keng 9 rice seedling shoots, cytokinin oxidase is more active in summer season than in winter. Cucumber seedling roots have strongest cytokinin oxidase activity in Dec, less in March and weak in May.
6. Acriflavin ( inhibitor of flavoproteins ) prohibits cytokinin oxidase activity of cytosol fraction, but didn’t inhibit that of cell wall fraction in corn seedling roots.
7. Cytokinin oxidase in rice seedling shoots of Tai-keng 3 and Tai-keng 9 exhibit optimal activity at pH 8.0 and 5mM CuCl2 of the reaction medium.

第一章 前言…………………………………………………… 1
第二章 文獻回顧……………………………………………… 2
（一） Cytokinin的生理功能………………………………… 2
（二） Cytokinin的種類與活性……………………………… 4
（三） Cytokinin在植物體內的合成………………………… 5
（四） Cytokinin在植物體內的代謝………………………… 7
1. 在adenine 環上做修飾……………………………… 7
2. 在N6側鏈上做修飾…………………………………… 8
（五） Cytokinin oxidase的功能…………………………… 12
（六） Cytokinin oxidase活性的調控……………………… 15
1. 內生及外加CK的含量………………………………… 15
2. auxin的濃度…………………………………………… 15
3. Phenolic化合物……………………………………… 16
4. phenylurea化合物…………………………………… 16
5. 自身glycosylation的影響…………………………… 17
6. isozyme的差異………………………………………… 17
（七） 蛋白質定量法………………………………………… 18
（八） Cytokinin oxidase的活性分析……………………… 19
1. 材料準備……………………………………………… 19
2. 試劑備製……………………………………………… 19
3. Cytokinin oxidase分析……………………………… 20
第三章 材料與方法…………………………………………… 21
（一） 實驗材料及生長培養之方法………………………… 21
（二） Cytokinin oxidase之粗萃取………………………. 22
（三） 細胞胞器之分離……………………………………… 24
（四） 蛋白質定量分析……………………………………… 25
1. 蛋白質試劑配製……………………………………… 25
2. 標準曲線製作………………………………………… 25
3. 蛋白質含量之測定…………………………………… 25
（五） Cytokinin oxidase定量分析………………………… 26
1. 試劑製備……………………………………………… 26
2. 標準曲線製作………………………………………… 26
3. 樣品中cytokinin oxidase活性檢測………………… 26
第四章 結果與討論…………………………………………… 28
（一） Cytokinin oxidase作用的溫度對其活性的影響…… 28
（二） Cytokinin oxidase 的活性在不同胞器中的表現… 29
（三） 不同成長天數對cytokinin oxidase 活性的影響… 30
（四） Cytokinin oxidase 的活性在根尖部位表現……… 31
（五） 季節變遷對cytokinin oxidase 活性的影響……… 31
（六） 抑制劑Acriflavin對cytokinin oxidase的影響…… 32
（七） pH值、CuCl2 對cytokinin oxidase的影響………… 33
參考文獻………………………………………………………….. 48

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