臺灣博碩士論文加值系統

本研究分成兩個部分：
第一部分探討以重組酪胺酸酶進行生物轉換。藉由普遍存在於豆科植物，並且容易大量取得的大豆異黃酮-糖基化daidzin和genistin作為基質，利用Escherichia coli表現來自Bacillus megaterium的酪胺酸酶，進行生物轉換。經過轉換反應後，兩個產物經由純化與NMR核磁共振鑑定，分別為3'-hydroxydaidzin和3'-hydroxygenistin。在自由基清除的實驗中，3'-hydroxydaidzin和3'-hydroxygenistin比起它們的前驅物daidzin和genistin，活性分別高出120和72倍。本研究是首度證實可應用酪胺酸酶生物轉換來生產3'-hydroxydaidzin和3'-hydroxygenistin，並顯示產物具有抗氧化活性，在產業上，應有高度實用與量產之潛力。
第二部分，以單細胞之模式生物衣藻(Chlamydomonas reinhardtii) 來探討nucleoredoxin (NRX)的功能。衣藻基因體中含有3種NRX基因，其確切之生理角色與分工仍然不清楚。先前實驗室研究發現由外源基因插入誘發之突變株HKCW19鞭毛異常短小，且其插入之突變位置為Nucleoredoxin 2 (NRX2)的3’UTR上。在本研究中，以RTq-PCR測量HKCW19之NRX2基因表現，卻發現與野生型CW15並無差異。再由衣藻突變種源庫獲得另一株突變株nrx2-1，發現其NRX2基因不表現，但含有正常鞭毛且會游動。因此，NRX2的缺失不會影響到鞭毛的生長。在nrx2-1突變株中，生長對數期時出現較高比例的大型細胞，且對過氧化氫的耐受度較差。進一步探討過氧化氫清除酵素catalase的基因轉錄、蛋白質表現與酵素活性，發現雖然mRNA與蛋白質的表現不受影響，但蛋白活性卻受到嚴重抑制。由此結果得知，衣藻的Nrx2功能類似於阿拉伯芥的NRX1，可調控catalase的活性。在nrx2-1細胞中，NRX2的缺失使NRX3 mRNA表現上升，而NRX1不受影響。另外，在野生型CC-5325中，經過pH shock逆境處理後，NRX2與NRX3的mRNA表現都會上升，但NRX1沒有明顯改變。這些基因表現的分析結果暗示NRX2與NRX3可能具有類似功能，或共同參與相同的生理反應。

This thesis comprises two parts to study two proteins responsible for redox reactions:
The first part describes the biotransformation of a commercially available rude extract of soy isoflavones by recombinant Escherichia coli expressing a tyrosinase from Bacillus megaterium. The original crude soy extracts contains significant amounts of the isoflavone glycosides daidzin and genistin. After biotransformation, two major products were isolated and identified as 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively. The free radical scavenging activities of the two 3'-hydroxyisoflavone glycosides were, respectively, higher than the activity of their precursors. This is the first demonstration of the bio-production and potential antioxidant applications of both 3'-hydroxydaidzin and 3'-hydroxygenistin.
In the second part, the unicellular organism Chlamydomonas reinhardtii was used to analyze the function of nucleoredoxin (NRX) genes. There are three members in the NRX family in Chlamydomonas, but their physiological roles and functional differentiation remains unclear. An exogenous DNA insertion was mapped to the 3’UTR of the NRX2 gene in a flagellum-defected mutant HKCW19, but RT-qPCR showed that the NRX2 transcript was not affected. In another nrx2-1 mutant, the NRX2 expression was abolished, but it contained normal flagella and can swim. Therefore, the disruption of NRX2 does not affect flagellar growth. The nrx2-1 mutant contained a higher ratio of large cells and was more sensitive to H2O2 treatment. The absence of NRX2 does not affect the transcript and protein levels of catalase (CAT), an enzyme responsible for H2O2 clearance. However, the activity of CAT is severely reduced in nrx2-1. Interestingly, loss of NRX2 caused the increase of NRX3 mRNA expression while NRX1 was not affected. Both NRX2 and NRX3 mRNA were induced after pH-shock treatment while NRX1 was not. These expression analyses suggest that NRX2 and NRX3 share similar functions or are involved in the same pathway.

摘要 i
Abstract ii
致謝 iv
目次 v
圖目次 ix
縮寫對照表 xi
第一篇 總論 1
第一章 緒論 2
1.1 研究目的 2
1.2 實驗架構 4
1.3 研究背景 2
第二章　文獻回顧 4
2.1 異黃酮 5
2.2 異黃酮多酚之生物活性 7
2.3 異黃酮多酚來源及其生產方式 8
第三章　材料與方法 13
3.1 材料 13
3.1.1藥品 13
3.1.2 Primers 序列及訂購公司 14
3.1.3溶液配製 15
3.1.4儀器 18
3.1.5菌株及細胞來源 19
3.2方法 20
3.2.1菌株培養與基因工程 20
3.2.1.1菌株培養 20
3.2.1.2菌株活化 20
3.2.1.3核酸(DNA)萃取 20
3.2.1.4質體(Plasmid)萃取 21
3.2.1.5聚合酵素連鎖反應(Polymerase chain reaction, PCR) 22
3.2.1.6膠體電泳(Electrophoresis) 23
3.2.1.7 DNA膠體純化(Gel extraction) 23
3.2.1.8基因選殖(Cloning) 24
3.2.1.9限制酶切割反應(Restriction enzyme digestion) 25
3.2.1.10質體轉形熱休克實驗(Heat shock) 25
3.2.1.11蛋白質電泳分析法 26
3.2.2生物轉換與產物純化與鑑定 26
3.2.2.1生物轉換 26
3.2.2.2超高效液相層析法( Ultra Performance Liquid Chromatography, UPLC ) 27
3.2.2.3濃縮萃取 27
3.2.2.4產物分離純化 28
3.2.2.5冷凍乾燥 28
3.2.2.6重組蛋白酪胺酸酶活性測試 28
3.2.2.7清除DPPH自由基能力之測定 29
3.2.2.8數據統計分析 29
第四章　實驗結果與討論 30
4.1 pETDuet-BmTYR載體建立 30
4.2 pETDuet-BmTYR蛋白質表現測試 31
4.3基因重組E.coli表現BmTYR活性測試 32
4.4基因重組E. coli進行D與G之生物轉換 33
4.5產物的分離純化及產物結構鑑定 34
4.6產物3'-OHD與3'-OHG對於DPPH自由基的清除能力 37
第五章　結論及未來展望 39
5.1結論 39
5.2未來展望 39
第三篇 衣藻Nucleoredoxin基因功能之初探 40
第一章 緒論 40
1.1研究背景 40
1.2研究目的 41
1.3實驗架構 42
第二章　文獻回顧 43
2.1模式生物-衣藻 43
2.1.1衣藻的結構與優勢 43
2.1.2衣藻突變株之生成 43
2.1.3分析外源基因插入於衣藻突變株之基因位置 44
2.2 Nucleoredoxin (NRX)蛋白之介紹 45
2.2.1 Nucleoredoxin之分類及結構 45
2.2.2 Nucleoredoxin調節的路徑與功能 47
第三章　材料與方法 50
3.1材料 50
3.1.1 Primers 序列及訂購公司 50
3.1.2溶液配製 51
3.1.3衣藻細胞來源 55
3.2方法 55
3.2.1藻株培養 55
3.2.2細胞計數 56
3.2.3核酸(DNA)萃取 56
3.2.4聚合酵素連鎖反應(Polymerase chain reaction, PCR) 57
3.2.5膠體電泳(Electrophoresis) 58
3.2.6 DNA膠體純化(Gel extraction) 58
3.2.7蛋白質電泳分析法 59
3.2.8西方墨點法(Western blot) 59
3.2.9 Spot test 60
3.2.10 RNA萃取 61
3.2.11 Reverse Transcription 反轉錄 62
3.2.12即時聚合酶鏈鎖反應( Real-time polymerase chain reaction, qPCR) 63
3.2.13製備衣藻細胞萃取液 64
3.2.14過氧化氫酶(Catalase)活性分析 65
3.2.15衣藻去鞭毛與鞭毛再生 65
3.2.16數據統計分析 66
第四章　實驗結果與討論 67
4.1外源基因插入衣藻鞭毛異常HKCW19之NRX2基因座 67
4.2在衣藻中，NRX2的缺失並不會影響鞭毛生合成 71
4.3在衣藻中，NRX2會調節抗氧化酵素的活性 77
4.4 在衣藻中，NRX2與NRX3可能參與類似的生化路徑 81
第五章　結論及未來展望 86
5.1結論 86
5.2未來展望 86
第四篇 總結 87
參考文獻 88
附錄一　pETDuet-1載體及cloning site 圖 97
附錄二　pETDuet-1 multiple cloning site圖 98
附錄三　BmTYR之基因序列 99
附錄四　pETDuet-BmTYR定序結果 100
論文著述 102

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