臺灣博碩士論文加值系統

染色體端節 ( telomere ) 是一種特殊的核蛋白構造，位於染色體的兩端，是維持基因組之完整性所必需，而染色體端節長度的變化在細胞的老化上扮演著重要的角色。染色體端節的維持須靠一種酵素，稱為端節酵素 ( telomerase )。它是一種核糖核酸蛋白酵素，屬於特殊的 DNA 聚合酵素，具修補能力，而在能永久增生的組織細胞中，皆有端節酵素的表現。因此，在分裂的植物細胞中，端節酵素的表現，能提供長期的增殖能力。本研究是以矮牽牛、菊花花苞、菊花小花、文心蘭及芥菜花序為對象，探討植物體於開花時期細胞內端節酵素之活性。結果顯示，矮牽牛瓶苗第一片展開葉雖然可以偵測得到端節酵素活性，但第一片展開葉中端節酵素之活性表現亦明顯地低於癒合組織。在菊花花苞方面，剛形成之花苞具有很強的端節酵素活性表現，之後端節酵素活性就開始下降，直至花苞即將要開放時，端節酵素活性就開始增加，所以端節酵素活性並非一直處於高度表現之狀態。由此結果亦可推測，在花芽形成至花朵開放過程中，並非一直處於細胞分裂之旺盛期，在花芽形成後，端節酵素活性降低，顯示細胞分裂暫時停歇，待花朵開始開放，細胞分裂再趨旺盛。菊花小花在 2 - 2.5 cm 時，具有最強之細胞分裂潛能，其端節酵素活性最高，且使用菊花花瓣進行癒合組織培養時，最先形成癒合組織，且生長情況最良好的，便是小花長度 2 - 2.5cm 者。但在文心蘭花朵開放之過程中，端節酵素活性之表現呈高低起伏之現象，且不曾降至幾乎完全缺乏的情況，只要端節酵素活性稍微下降後，似乎有立即補充的機制，端節酵素活性便又立刻上升。芥菜花序端節酵素活性表現情形和文心蘭的結果相去不遠。芥菜花可能因其再生能力很強，花序被摘除後，很快又能長出新的成串花芽，具有很強的生長勢。菊花小花染色體端節平均長度介於 6.6 kb-16.2 kb，小花長度為 2 - 2.5 cm時染色體端節長度最長，約為 16.2 kb，在此時，其端節酵素之活性表現亦為最高，顯示端節酵素之活性表現與端節 DNA 長度成正相關。

Telomeres are nucleoprotein structure at the ends of linear eukaryotic chromosomes that are essential for maintaining the integrity of the genome. The changes in telomere length may play an important role in the senescence of cells. Telomere is maintained by a special enzyme named telomerase. It is a ribonucleoprotein with a reverse transcriptase activity . Telomerase is a specialized DNA polymerase which is present in regenerating tissues. Hence , telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity. We studied the telomerase activities of petunia, Chrysanthemum floral buds , Chrysanthemum florets , Oncidium and Brassica juncea inflorescence during flowering stage. In petunia , telomerase activity could be detected in the cultured primary leaves , but the expression is lower than callus. In Chrysanthemum floral buds , the young buds have strong telomerase activity expression , then this activity decrease. Telomerase activity increase until flowering. Apparently it is not stay at high level all the time in flower. In Chrysanthemum florets , the 2-2.5cm lengh have the highest telomerase activity expression and strongest cell division potential. When cultures of Chrysanthemum florets were initiated, the finest growth situation was appeared to be the lengh between 2-2.5cm. In Oncidium, it seems to have a resupply mechanism, for telomerase activity because its expression, even little, always exist. A similer result was obtained with Brassica juncea. Maybe due to high regenerating capacity in Brassica juncea. The telomere lenghs in Chrysanthemum florets were between 6.6 kb - 16.2 kb . The 2 -2.5 cm Chrysanthemum florets have the longest telomere length as well as the highest telomerase activity.

中文摘要……………………………………………………………..i
英文摘要……………………………………………………………..iii
目錄…………………………………………………………………..iv
壹、 前言…………………………………………………………….1
貳、 前人研究……………………………………………………….4
一、 染色體端節與染色體端節酵素…………………………4
二、 染色體端節酵素與癌症的關係…………………………9
三、 染色體端節酵素與細胞老化的關係……………………12
四、 植物老化…………………………………………………14
五、 植物體的染色體端節……………………………………16
六、 端節酵素於植物體的表現………………………………17
參、 實驗材料與方法……………………………………………….24
一、 植物材料…………………………………………………24
二、 細胞萃取液 ( cell lysate ) 的製備…………………………25
三、 細胞萃取液蛋白質定量分析……………………………28
四、 端節酵素活性之偵測……………………………………29
五、 染色體端節長度 ( telomere lengh ) 的測量…………….34
六、 活性分析方法……………………………………………..40
肆、 結果……………………………………………………………..42
一、 細胞萃取液蛋白質定量分析……………………………42
二、 端節酵素活性之偵測……………………………………42
三、 染色體端節長度的測量…………………………………56
伍、討論……………………………………………………………..62
一、 細胞萃取液蛋白質定量分析……………………………62
二、 染色體端節長度的測量…………………………………67
陸、參考文獻………………………………………………..………69

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