臺灣博碩士論文加值系統

β-澱粉酶 (β-amylase，EC 3.2.1.2) 屬於一種外切苷酶 (exoglycosidase)的特性已為人熟知，然而其生理意義尚未明確瞭解。綜合以前的文獻結果皆顯示，逆境誘導β-澱粉酶活性必須要有光照。由於前人研究的光源都是以全光為主，因此本論文進一步利用發光二極體 (Light Emitting Diode; LED) 提供不同的光質條件，探討不同光質對甘藷癒創組織表現β-澱粉酶的影響，同時透過不同試劑的處理，探討誘導β-澱粉酶表現的訊息傳遞機制及其生理意義。
甘藷癒創組織在藍光照射下，β-澱粉酶活性的表現明顯高於紅光或遠紅光處理。同時給予藍光及滲透逆境 (8% w/v,蔗糖)處理，對甘藷細胞表現β-澱粉酶活性有顯著的加成效應。同樣地，RT-PCR、酵素活性分析、膠體活性染色及西方墨點法的經時變化結果得知，藍光及滲透逆境誘導β-澱粉酶的表現皆顯著高於黑暗處理，並伴隨麥芽糖的累積。由於文獻指出，麥芽糖具有可親和溶質的特性，因此合理推論β-澱粉酶可能具有防衛性功能的角色。由於 cycloheximide (100 μM)可以抑制80%以上的β-澱粉酶活性，因此藍光誘導甘藷癒創組織累積β-澱粉酶可能是透過重新合成。外加 NADPH 氧化酶抑制劑– diphenylene iodonium (2 μM) 確實減少細胞內過氧化氫 (hydrogen peroxide, H2O2)的含量，同時抑制藍光誘導β-澱粉酶70%的表現。經時變化的結果除了顯示 H2O2 的含量變化與β-澱粉酶轉錄表現有一致性外，藍光處理的細胞在抗氧化相關酵素，例如 superoxide dismutase (EC 1.15.1.1)、catalase (EC 1.11.1.6)及 peroxidase (EC 1.11.1.7)的表現亦明顯高於黑暗處理。因此，甘藷細胞在藍光照射下透過 H2O2 活化β-澱粉酶的表現。由於不論提高光照頻率 (從60至180 Hz)或光照強度 (從15至75 μmole-2s-1)並不會改變β-澱粉酶的表現。β-澱粉酶的表現不受 DCMU (2 μM)的影響，但 methyl viologen (2 μM)則明顯抑制，因此推測藍光可能僅是一種訊息，而且藍光誘導甘藷癒創組織表現β-澱粉酶可能與光合系統Ⅰ的作用有關。
雖然茉莉酸與水楊酸皆屬於防禦性荷爾蒙，然而β-澱粉酶的表現會受到水楊酸抑制;相對地，茉莉酸則顯著增加細胞內的 H2O2 含量及表現β-澱粉酶。此結果除了進一步確認 H2O2 與甘藷癒創組織表現β-澱粉酶的相關性以及β-澱粉酶具有防禦性蛋白的功能。

β-Amylase (EC 3.2.1.2) is well-known to the activity of exoglycosidases; however, its true physiological significance is not completely known. According to the results of previous reports, stress-induced β-amylase was dependent on light. Due to full light was adopted in previous studies, in this thesis, the specific light quality supported by light emitting diode was used to investigate the effect of light quality on the expression of β-amylase of sweet potato callus. Meanwhile, the pharmacological study was also used to deduce the signal transduction pathway about β-amylase induction and its physiological significance.
Under osmotic stress [8% (w/v), sucrose] condition, the calli exposed to blue light (30 μmole m-2s-1) showed the activity of β-amylase was significantly higher than that of red and far-red light irradiation. Compared with dark treatment, the results of time course analysis, such as RT-PCR, activity assay, Native-PAGE staining and Western blot, all showed that blue light stimulated β-amylase expression at the transcriptional and translational levels, then, caused the blue light-grown calli had a higher maltose content. Maltose has been reported to have compatible solute properties, it is reasonable to deduce that β-amylase may have role in the defense function. Since the activity of β-amylase was inhibited by cycloheximide (100 μM) over 80%, suggesting that blue light induced β-amylase was via a de novo synthesis. Exogenous diphenylene iodonium (2 μM)‐ a inhibitor of NADPH oxidase, actually decreased the amount of hydrogen peroxide (H2O2) and simultaneously inhibited ca. 70% of the β-amylase activity. The changes of H2O2 content were also consistent with the expression of β-amylase in transcription level. Moreover, the activities of antioxidative-related enzymes, such as superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and peroxidase (EC 1.11.1.7) in blue light condition all were stimulated. Hence, H2O2 seemingly acts as a mediator for the activation of β-amylase in sweet potato callus under blue light irradiation. In addition, the activity of β-amylase did not effect by rising the light frequency (from 60 to 180 Hz) or intensity (from 15 to 75 μmole-2s-1), and by DCMU (2 μM), but it was obviously inhibited by methyl viologen (2 μM). These data strongly imply that blue light just acts as a signal to induce the expression of β-amylase in sweet potato callus, and this inductive phenomenon is closely correlated with potosystem I.
Jasmonic acid (JA) and salicylic acid (SA) both belong to a defensive hormone. Salicylic acid inhibited the expression of β-amylase in sweet potato cells. By contrast, JA not merely increased β-amylase activity, but also enhanced H2O2 level. These results further confirm that β-amylase plays a defensive role and its expression is linked with H2O2.

頁次
謝誌------------------------------------------------------------------I
目錄------------------------------------------------------------------II
表目錄----------------------------------------------------------------Ⅴ
圖目錄----------------------------------------------------------------Ⅵ
縮寫表----------------------------------------------------------------Ⅷ
中文摘要---------------------------------------------------------------1
英文摘要---------------------------------------------------------------2
第一章 前言 -----------------------------------------------------------4
1.1 甘藷的簡介---------------------------------------------------------4
1.2 文獻回顧-----------------------------------------------------------5
1.2.1 植物中β-澱粉酶的作用與角色--------------------------------------5
1.2.2 不同光質對植物生長與生理方面的影響------------------------------7
1.2.3 發光二極體的基本原理及在植物上的應用-----------------------------7
1.2.4 植物的防禦機制與調控---------------------------------------------9
1.3 研究動機與目的----------------------------------------------------15
第二章 材料與方法 ----------------------------------------------------16
2.1 實驗材料----------------------------------------------------------16
2.2 試劑處理----------------------------------------------------------16
2.3 甘藷癒創組織之β-澱粉酶的分析--------------------------------------17
2.3.1 酵素液的萃取---------------------------------------------------17
2.3.2 β-澱粉酶活性分析-----------------------------------------------17
2.3.2.1 試管分析----------------------------------------------------17
2.3.2.2 Native-PAGE 膠體電泳活性分析--------------------------------18
2.3.3 西方墨點法-----------------------------------------------------19
2.4 甘藷癒創組織之 RT-PCR 分析----------------------------------------21
2.4.1 Total RNA 的萃取-----------------------------------------------21
2.4.2 反轉錄聚合連鎖反應---------------------------------------------23
2.5 抗氧化酵素活性分析------------------------------------------------26
2.5.1 超氧歧化酶之萃取與活性分析-------------------------------------26
2.5.2 Catalase 之萃取與活性分析酵素活性分析--------------------------28
2.5.3 過氧化酶之萃取與活性分析---------------------------------------28
2.6 蛋白質定量--------------------------------------------------------29
2.7 甘藷癒創組織之 H2O2 含量測定--------------------------------------30
2.8 碳水化合物分析----------------------------------------------------31
2.8.1 可溶性醣的萃取-------------------------------------------------31
2.8.2 醣類成份的分析-------------------------------------------------32
第三章 結果 ----------------------------------------------------------37
3.1 不同光質誘導甘藷癒創組織表現β-澱粉酶的影響------------------------37
3.2 藍光及滲透逆境對甘藷癒創組織生長及β-澱粉酶表現的影響--------------37
3.3 不同試劑處理對甘藷癒創組織表現β-澱粉酶的影響----------------------39
3.4 活氧化物對甘藷癒創組織表現β-澱粉酶的影響--------------------------40
3.5 抗氧化相關酵素活性分析--------------------------------------------40
3.6 外加 JA 及 SA 對甘藷癒創組織表現β-澱粉酶的影響--------------------41
第四章 討論-----------------------------------------------------------62
第五章 結論-----------------------------------------------------------65
第六章 參考文獻-------------------------------------------------------67

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