臺灣博碩士論文加值系統

自農委會水產試驗所東港分所取得的螺旋藻及綠球藻，利用光學顯微鏡僅能初步觀察其型態為螺旋狀及球狀藻體，配合穿透式電子顯微鏡更加確認其內部結構，發現螺旋藻具有明顯的septa、類囊體及不甚明顯的核區，綠球藻則具有纖毛、澱粉核及類囊體等。比對螺旋藻及綠球藻的核糖體DNA，發現螺旋藻與GenBank 資料庫中Arthrospira maxima (AJ292323)有100% 相似度，而綠球藻與Nannochloris及Chlorella相似度約71-76%。
在DPPH自由基清除之抗氧化能力測試方面，螺旋藻及綠球藻和標準品VitE、BHA與相同濃度甲醇萃取液0.4 mg/ml作比較時，兩藻種抗氧化能力約為VitE和BHA的一半，抗氧化能力大小依序為α-tocopherol > BHA > Chlorella sp. > Arthrospira sp.。當兩藻種濃度提高至1.2 mg/mL時，抗氧化能力提高至90%，與標準品VitE、BHA非常相近，此時抗氧化能力達到高峰且維持穩定。另經DEAE陰離子交換管柱層析純化之超氧歧化酵素(SOD)，螺旋藻的SOD比活性為875 U/mg，為原先初萃取物的18.3倍；綠球藻則為960 U/mg為原先初萃取物的17.9倍。此結果與靈芝及金針菇相比較下，兩藻種之SOD活性約高三倍，顯示螺旋藻及綠球藻確定具有很高的SOD活性，可作為以抗氧化特性為訴求的生藥保健產品。

Using a light microscope to study Spirulina sp. or Chlorella sp., which were obtained from the Marine Products Research Institute at Tungkang, Pingting County, only its external morphology, as spiral or round shape, could be observed. However, when supplemented by a transmission electron microscope, its internal structures could be determined, showing septa, thylacoid and a not-well-defined nucleus in Spirulina sp. and cilia, pyrenoid and thylacoid in Chlorella sp. When comparing rDNA of Spirulina sp. and Chlorella sp. with those deposited in GenBank, the results showed that there was 100% similarity between Spirulina sp. and Arthrospira maxima (AJ292323) and about 71-76% similarity between Chlorella sp. and Nannochloris sp.
The study on the ability of the free radical DPPH in eliminating antioxidation showed that, when comparing A. maxima and Chlorella sp. with the standards VitE and BHA under the same concentration of 0.4mg/ml, the antioxidative activities of these two algae were about half of that of VitE and BHA. The antioxidative activities were arranged on the basis of their intensity, in a descending order, VitE>BHA>Chlorella sp. >A. maxima. When the concentration was increased to 1.2 mg/ml, the antioxidative activities of A. maxima and Chlorella sp. increased to about 90%, which was close to that of VitE and BHA. That intensity was at its peak at this concentration and remained so in a stable condition. After purifying through the DEAE anion exchange column, the activities of the superoxide dismutase (SOD) of A. maxima. were 875U/mg, which was 18.3 times more than that of its initial extracts. The same activities of Chlorella sp. were 960U/mg, about 17.9 times of its initial extracts. This result showed that the SOD’s of these two algae were three times higher than that of Ganoderma lucidum and mushroom, demonstrating that there were high SOD activities in A. maxima and Chlorella sp. A. maxima and Chlorella sp., therefore, have a high potential for developing into pharmaceutical or health-related products, emphasizing as having specialized antioxidative characteristics.

目 錄
中文摘要………………………………………..………………….I
英文摘要……………………………………...………………….III
誌謝…………………………………...…………………..…….…V
目錄…………………………………...…………………...….…VII
圖次…………………………………...…………….……...…….XI
表次……………………………………...………………………XV
第一章 緒言………………………..……………………………1
第二章 文獻探討…………………………...…………...………5
2.1自由基與活性氧的形成…………...………...……………5
2.2自由基與活性氧對細胞的影響…………………………..7
2.2.1自由基與活性氧對細胞的損傷……...……………9
2.2.2自由基與活性氧對生命的損傷…………….……11
2.2.3自由基與活性氧對酵素活性的破壞…….....……11
2.2.4自由基與活性氧對核酸的破壞……...……..……11
2.3細胞的抗氧化作用…………………...………………….12
2.3.1 超氧歧化酵素之抗氧化…………..……….……15
2.3.2 SOD的生理機能…………………….……….…..18
2.4螺旋藻、綠球藻抗氧化的研究…………….……….……19
第三章 材料與方法………………………...………………….23
3.1藻種培養……………………….………...……………....23
3.2藻種鑑定……………………….……...…………………25
3.2.1光學顯微鏡觀察…………….……………………25
3.2.2穿透式電子顯微鏡…………….…………………25
3.2.3螺旋藻及綠球藻生化反應測定………….………26
3.3核糖體核酸序列分析…………………...………………26
3.3.1藻類基因組的抽取……………………………….26
3.3.2聚合酵素連鎖應………...….…………..………...27
3.3.3接合作用………………………………………….28
3.3.4轉型作用……………………………...….…….…28
3.3.5小量質體DNA抽取及重組確認………….….….29
3.3.6質體的純化……………………………...………..30
3.3.7核酸的定序與分析……………………………….30
3.4 清除自由基α,α-diphenyl-β-picrylhydrazyl (DPPH)抗氧化能力測定…………………………………….31
3.5 超氧歧化酵素SOD萃取鑑定與純化…………...……..31
3.5.1粗酵素液的製備……………………….…………31
3.5.2酵素之蛋白質含量測定………………….………32
3.5.3 酵素之SDS-PAGE電泳分離…………...………33
3.5.4超氧歧化酵素活性染色…………...……………..33
3.5.5 超氧歧化酵素活性測試(NBT的光還原反應法)
………………………………………………….34
3.5.6 超氧歧化酵素純化……………………………...34第四章 結果…………………………………...………………..36
4.1基本遺傳形質鑑定………………………...…………….36
4.1.1螺旋藻形態微細構造……………………...……..36
4.1.2綠球藻形態微細構造……………………...……..37
4.2螺旋藻及綠球藻之生化反應探討…………...……37
4.3核酸的定序與分析……………………...…………37
4.4 DPPH抗氧能力測定…………………...…………39
4.5超氧歧化酵素純化及活性測定…………...………40
4.6 超氧歧化酵素型態鑑定(SDS-PAGE and
Native-PAGE)……………………………………40
第五章 討論…………………………...……………...…….…..42
第六章 結論…………………………...…………...……….…79
參考文獻 ………………………...…………………..…………..80
附錄一 選殖載體pGEM-T Easy Vector 圖譜………….…..…88
附錄二 選殖載體pGEM-T Easy Vector限制酶切割位……….89
作者簡介…………………...………………………..……………90
圖 次
頁 次
圖2-1穩定形態之三重態氧(3O2)形成各種反應性分子
(1O2, O-2,H2O2及HO.)過程……………..………………... 6
圖2-2生物體內抵抗自由基機制………………..………….….…8
圖2-3自由基對細胞的影響…………..………………………....10
圖3-1綠球藻及螺旋藻的培養情形…..………………………....46
圖3-2螺旋藻的培養情形…………………………………..…... 46
圖 3-3螺旋藻Arthrospira maxima葉綠體(chloroplast)之rDNA
、tRNA基因位置圖………………..……………………..47
圖3-4綠球藻(Chlorella sp.)細胞核(nucleus)之rDNAs及內轉錄區(ITS)基因位置圖…………………...…..……….....….48
圖3-5 SOD活性對反應吸光值的關係…………..……...………49
圖3-6不同活性的SOD對NBT光還原反應之抑制率…………50
圖4-1光學顯微鏡下的螺旋藻(200X)……...…………...……….51
圖4-2經DAPI染色後螢光顯顯微鏡下的螺旋藻(600x)……….51
圖4-3光學顯微鏡下的綠球藻(400X)…………………………...52
圖4-4經DAPI染色後螢光顯微鏡下的綠球藻(600x)……….....52
圖4-5螺旋藻(Arthrospira maxima)之穿透式顯微鏡照80,000X(a,c)………...…………………………………....53
圖4-6綠球藻(Chlorella sp.)之穿透式顯微鏡照...……...………54
圖4-7螺旋藻(Arthrospira maxima)、綠球藻(Chlorella sp.) genomic DNA聚合酵素連鎖反應產物之電泳分析……55
圖4-8使用primer A and primer B增幅螺旋藻(Arthrospir maxima)葉綠體之rDNA 、tRNA-Ile gene、 tRNA-Ala 基因列……………………………...………………….....56
圖4-9使用primer NS7及 ITS 4增幅綠球藻(chlorella sp.)之細胞核rDNA、內轉錄區(ITS)基因序列圖.……….…...….57
圖4-10螺旋藻(Arthrospira maxima)及綠球藻(Chlorella sp.)甲醇萃取物對DPPH自由基清除之反應動力模式……........58
圖4-11螺旋藻(Arthrospira maxima)及綠球藻(Chlorella sp.)甲醇萃取物對DPPH自由基清除之功效……………..….….59
圖4-12不同濃度甲醇萃取物的螺旋藻(Arthrospira maxima)及綠球藻(Chlorella sp.)對DPPH自由基清除能力…………60
圖4-13綠球藻(Chlorella sp.)之DEAE離子交換樹脂管柱層析…………………………………………………………61
圖4-14螺旋藻(Arthrospira maxima)之DEAE離子交換樹脂管柱層析……...……………………………………...……..…62
圖4-15螺旋藻(Arthrospira maxima)及綠球藻(Chlorella sp.)粗蛋白質液經SDS-PAGE變性解離之電泳分析……….…..63
圖4-16螺旋藻(Arthrospira maxima)及綠球藻(Chlorella sp.)粗蛋白質經DEAE純化後之活性染色電泳分析……………64
圖5-1 螺旋藻(Arthrospira maxima) 之部分16S rDNA、tRNA-Ile、tRNA-Ala、ITS與部份23S rDNA之基因序列與GenBank中四種螺旋藻相同基因區之序列比對...67
圖5-2 螺旋藻(Arthrospira maxima) 之部分16S rDNA、tRNA-Ile、tRNA-Ala、ITS與部份23S rDNA之基因序列與GenBank中四種螺旋藻相同基因區序列比對之親源關係樹狀圖………………………………………....…68
圖5-3 綠球藻(Chlorella sp.) 之部分18S rDNA 之基因序列與GenBank中四種綠球藻及兩種綠藻之相同基因區之序
列比對….………………………………….……………...71
圖5-4 綠球藻(Chlorella sp.) 之部分18S rDNA 之基因序列與GenBank中四種綠球藻及兩種綠藻相同基因區序列比
對之親源關係樹狀圖...……….………………………….72
表 次
頁 次
表2-1自由基參與的各種疾病…………………..………………14
表2-2一般超氧岐化酵素之種類及特性…..………..……..……17
表2-3 BG-11培養液的配製…...…………………………………24
表4-1綠球藻(Chlorella sp.)及螺旋藻(Arthrospira maxima)與其他綠球藻之生化特性比較……………...…………..……73
表4-2綠球藻(Chlorella sp.) 蛋白質各純化步驟回收率及純化倍率與SOD活性……………….………………...……..…..74
表4-3螺旋藻(Arthrospira maxima) 蛋白質各純化步驟回收率及純化倍率與SOD活性……………………...……..…...75
表 5-1螺旋藻(Arthrospira maxima) 之部分16S rDNA、tRNA-Ile、tRNA-Ala、ITS與部份23S rDNA之基因序列與GenBank中四種螺旋藻相同基因區之序列比對基因序列的相似度百分比………………………………....….76
表 5-2 綠球藻(Chlorella sp.) 之部分18S rDNA 之基因序列與
GenBank中四種綠球藻及兩種綠藻相同基因區序列比對之相似度百分比……………….…………...….……77
表 5-3 綠球藻(Chlorella sp.)及螺旋藻(Arthrospira maxima)，
靈芝(G. lucidum )，金針菇超氧歧化酵素活性比較………….………………………………………...….78

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