臺灣博碩士論文加值系統

真菌是一群多種型態的細胞，由菌絲組成的真核生物，其細胞壁構造與植物有明顯的差異，但缺乏葉綠素且無法行光合作用，屬異營性生物，其價值來自於二次代謝產物，到目前為止，此化合物應用於許多抗腫瘤、抗菌、及抗毒性的藥物，因此，在現今已被作為食品工業與醫藥用材之用途。過去文獻證實，真菌Neosartorya fischeri合成二次代謝產物的基因序列，有16組PKS與18組NRPS基因序列所調控。然而在過去研究顯示，Neosartorya fischeri所產生的二次代謝產物，目前只有三種化合物，代表尚有許多二次代謝產物還未被分離及鑑定。而在一般的實驗室培養條件下，Neosartorya fischeri有許多微量的二次代謝產物不容易被觀察，因此，我
們想要嘗試以分子生物的技術或是透過不同配方的培養基獲得二次代謝產物。透過Fusion PCR技術，結果顯示，將Neosartorya fischeri NRRL181基因串進行融合，目前WA5’/NFIA_043640與NFIA_043650/NFIA_043660片段已融合完畢。而另一方面透過培養天數得知，培養20天二次代謝物訊號能增加，而透過不同配方的培養基，改變環境因子pH值，結果顯示，pH值越呈鹼性，20和22分鐘的二次代謝產物訊號，會明顯的增強;而環境因子滲透壓，結果顯示，當鹽濃度越來越高時，主要的20和22分鐘的二次代謝產物訊號，會越來越低。期望能透過Fusion PCR技術，將Neosartorya fischeri NRRL181基因串進行融合，送入帶有nkuAΔ這段可刪除基因的Aspergillus nidulans表現系統中;以及藉由HPLC觀察，進一步獲取大量的二次代謝產物並建立資料庫，以利未來在臨床醫學或藥物治療
能有所貢獻或新的發展。

Fungi are a group of a variety of cell types, by the mycelium of
eukaryotes, there are significant differences in their cell wall structure and plant, but the lack of chlorophyll and not conducted photosynthesis, belonging to the abnormal Camp biological, its value comes from the secondary metabolites, far the way, Application of many anti-tumor, anti-bacterial and anti-toxicity drugs, therefore, as has been the use of food industry and pharmaceutical materials. Literature confirmed that Neosartorya fischeri synthetic gene sequence of secondary metabolites, there are 16 groups of PKS and 18 groups NRPS gene sequence of the regulation. However, studies have shown that in the past, secondary metabolites produced by Neosartorya fischeri, currently only three kinds of compounds, representative there are many secondary metabolites has not yet been isolated and identification. In laboratory culture conditions, Neosartorya fischeri has trace amounts of secondary metabolites is not easy to be observed, therefore, we want to try to molecular biological techniques, or through the medium of different formulations of secondary metabolites.By Fusion PCR, The results showed that fusion of Neosartorya fischeri NRRL181
gene string, the WA5''/NFIA_043640 and NFIA_043650/NFIA_043660 fragment fusion is complete. On the other hand through learned through training days, training 20 days secondary metabolite signals can increase; through the medium of different formulations, changing environmental factors pH value, the results showed that pH values more alkaline, 20 and 22 minutes of secondary metabolites signal will be significantly enhanced;Osmotic pressure of environmental factors, results showed that, when the salt concentration is getting higher and higher, 20 and 22 minutes of the main secondary metabolites signal will be getting lower and lower. Expectations through Fusion PCR technique,fusion of Neosartorya fischeri NRRL181gene string, transformation with carry nkuAΔ delete the genes, of Aspergillus nidulans expression system, obtain many of secondary metabolites;And observed by HPLC, and further access to a large number of secondary metabolites and to create a database to facilitate future in clinical medicine or drug therapy can contribute to new
development.

中文摘要……………………………………………………………………….Ⅰ
英文摘要……………………………………………………………………….Ⅲ
本文目錄……………………………………………………………………….Ⅴ表目錄………………………………………………………………………….Ⅸ
圖目錄…………………………………………………………………………. X
英文縮寫對表…………………………………………………………………XII
第一章 緒論…………………………………………………………………….1
1.1 真菌簡介………………………………………………………………1
1.2 真菌真菌的代謝產物…………………………………………………3
1.3 二次代謝產物合成酶…………………………………………………3
1.4 影響真菌Neosartorya fischeri的生長因子………………………….5
1.5 Neosartorya fischeri…………………………………………………...5
1.6 融合PCR……………………………………………………………...6
1.7 Aspergillus nidulans…………………………………………………...7
1.8 研究動機………………………………………………………………8
第二章 材料與方法…………………………………………………………...10
2.1實驗材料………………………………………………………………….10
2.1.1實驗菌株………….............................................................................10
2.1.2實驗藥品………………....................................................................10
2.1.3實驗儀器.............................................................................................10
2.2真菌之培養...............................................................................................12
2.2.1真菌固態培養條件…………………………………........................12
2.2.2真菌固態收集孢子……….…………………………………………12
2.2.3真菌液態培養條件………………………………………………….12
2.2.4真菌液態收集孢子………………………...……………………….13
2.2.5探討各因子對真菌Neosartorya fischeri生長之影響…………….13
2.2.5.1探討pH值固態培養對N. fischeri生長之影響…………............13
2.2.5.2探討鹽類(NaCl)固態培養對N. fischeri生長之影響....................13
2.3真菌genomic DNA之萃取……………………………………………..14
2.4 DNA濃度及純度測定……………………………………………..........15
2.5聚合酶鏈鎖反應………………………………………………..……….15
2.5.1融合PCR…………………………………………………………....16
2.5.2 Neosartorya fischeri NRRL181基因串各基因之引子…..............16
2.5.3 Neosartorya fischeri NRRL181基因串各基因擴增之引子:.........17
2.5.4Neosartorya fischeri NRRL181基因串2基因融合之引子………19
2.5.5 Neosartorya fischeri NRRL181基因串2基因融合擴增之子…..20
2.6瓊脂膠體電泳法…………………………………………………………21
2.6.1DNA亮帶膠體純化…………………………………………………22
2.7限制片段的分析……………………………………………………........23
2.8高效液相層析儀……………………………………………………........23
2.8.1樣品之萃取配方…………………………………………………....23
2.8.2樣品之萃取製備…………………………………………………....23
2.8.3樣品HPLC分析條件…………………………………………….......24
第三章 結果…………………………………………………………………...25
3.1 Neosartorya fischeri之培養………………..…………………..……..25
3.2 Neosartorya fischeri pH值及滲透壓之培養……......………………..25
3.3 Neosartorya fischeri之genomic DNA萃取…………………..……..26
3.4 Neosartorya fischeri NRRL181之基因串個別片段……………….26
3.5以限制酶酵素確認Neosartorya fischeri NRRL181之基因串個別片
段大小………………………………………………………………...27
3.6 Neosartorya fischeri NRRL181之基因串WA5’+NFIA_043640與
NFIA_043650/ NFIA_043660片段融合…………………………......27
3.7以限制酶酵素確認Neosartorya fischeri NRRL181之基因串WA5’/
NFIA_043640與NFIA_043650/ NFIA_043660之融合片段…........28
3.8真菌Neosartorya fischeri培養5天之二次代謝產物……………….28

3.9真菌Neosartorya fischeri培養10天之二次代謝產物………………29
3.10真菌Neosartorya fischeri培養15天之二次代謝產物…………….30
3.11真菌Neosartorya fischeri培養20天之二次代謝產物…………….30
3.12培養天數為5、10、15及20天之統計圖…………….…………….31
3.13真菌Neosartorya fischeri pH值為5.5之二次代謝產物…………..31
3.14 真菌Neosartorya fischeri pH值為7.5之二次代謝產物………….32
3.15 真菌Neosartorya fischeri pH值為9.5之二次代謝產物…………..32
3.16 環境因子pH值為 5.5、7.5及9.5之統計圖…………..………….33
3.17真菌Neosartorya fischeri NaCl為0%之二次代謝產物……………33
3.18真菌Neosartorya fischeri NaCl為1 %之二次代謝產物…………..33
3.19真菌Neosartorya fischeri NaCl為2.5 %之二次代謝產物…………34
3.20真菌Neosartorya fischeri NaCl為5 %之二次代謝產物…………...35
3.21環境因子NaCl為1、2.5及5%之統計圖………………………….35
第四章 討論…………………………………………………………………...36
第五章 結論…………………………………………………………………...40
參考文獻……………………………………………………………………….41
附圖…………………………………………………………………………….47

表目錄
表2-1實驗所使用之藥品……………………………………………………...10
表2-2實驗所使用之儀器……………………………………………………...11
表2-3實驗HPLC分析條件……………………………………………………24















圖目錄
圖 2-1Neosartorya fischeri NRRL181基因串之示意圖................................15
圖3-1 Neosartorya fischeri固態、液態、顯微鏡之觀察................................47
圖3-2培養Neosartorya fischeri pH值及滲透壓之20天……........................48
圖3-3 Neosartorya fischeri之genomic DNA萃取...........................................49
圖3-4 Neosartorya fischeri NRRL181之基因串個別片段...........................50
圖3-5 Neosartorya fischeri NRRL181之基因串個別片段之確認................51
圖3-6 Neosartorya fischeri NRRL181之基因串WA5’+NFIA_043640與
NFIA_043650/ NFIA_043660片段融合..................................................52
圖3-7以限制酶酵素確認Neosartorya fischeri NRRL181之基因串WA5’/
NFIA_043640與NFIA_043650/ NFIA_043660之融合片段……….....53
圖3-8以HPLC分離真菌Neosartorya fischeri 培養5天之圖譜…………..54
圖3-9以HPLC分離真菌Neosartorya fischeri 培養10天之圖譜…………55
圖3-10以HPLC分離真菌Neosartorya fischeri 培養15天之圖譜………..56
圖3-11以HPLC分離真菌Neosartorya fischeri 培養20天之圖譜………..57
圖3-12培養天數為5、10、15及20天之統計圖…………………………..58
圖3-13 以HPLC分離真菌Neosartorya fischeri pH值為5.5之圖譜………59
圖3-14 以HPLC分離真菌Neosartorya fischeri pH值為7.5之圖譜………60
圖3-15以HPLC分離真菌Neosartorya fischeri pH值為9.5之圖譜………61
圖3-16 環境因子pH值為 5.5、7.5及9.5之統計圖………........................62
圖3-17以HPLC分離真菌Neosartorya fischeri NaCl為0%之圖譜……….63
圖3-18 以HPLC分離真菌Neosartorya fischeri NaCl為1%之圖譜………64
圖3-19 以HPLC分離真菌Neosartorya fischeri NaCl為2.5%之圖譜…….65
圖3-20 以HPLC分離真菌Neosartorya fischeri NaCl為5%之圖譜………66
圖3-21 環境因子NaCl為0、1、2.5及5%之統計圖………………………67

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