Neosartorya fischeri NRRL 181基因的選殖與二次代謝產物的探討

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Title:	Neosartorya fischeri NRRL 181基因的選殖與二次代謝產物的探討 Investigation of Neosartorya fischeri NRRL 181 gene cloning and secondary metabolites prodution
Authors:	許敬琳
Contributors:	生物科技系暨研究所洪瑞祥
Keywords:	二次代謝產物碳源微量元素 secondary metabolites fusion PCR sourcetrace elements Carbon Aspergillus nidulans(nkuAΔ) Neosartorya fischer
Date:	2012
Issue Date:	2012-11-29 11:35:48 (UTC+8)
Abstract:	真菌的二次代謝產物是農業和醫療所需的生物活性化合物的重要的來源。根據文獻表示，在1993與2001年期間分離和鑑定發現1500種的二次代謝物有一半以上含抗細菌、抗真菌、抗腫瘤的活性。並過去的研究發現，真菌二次代謝產物主要由聚酮合成酶（PKS）和非核糖體肽類合成酶（NRPS）所合成的。在以往的資料指出，Neosartorya fischeri此株菌預測含有16組PKS和18組NRPS的基因簇來合成二次代謝產物。目前研究Neosartorya fischeri二次代謝物相關的文獻非常的稀少，且從Neosartorya fischeri的基因組預測已顯示，還有大量的二次代謝產物尚未發覺。這可能是因為許多次二次代謝產物基因簇在正常的實驗室培養條件下是不會表現的。因此，我們要使用分子生物技術來表達這些基因。目前本實驗使用融合PCR完成了其中NFIA_043670融合基因，與NFIA_043680、NFIA_043690和PyroA/WA3’的選殖。並在實驗中又更換不同的碳源來探討二次代謝物的表現，利用葡萄糖作為控制組的碳源，以單醣中的木糖、果糖與雙糖中的蔗糖與乳糖來作為實驗組，且高效液相層析偵測發現，以碳源為木糖其曲線下面積表現最佳，葡萄糖、果糖、蔗糖次之，而乳糖表現出來曲線下面積最差，本實驗又添加氯化銨作為氮源發現對二次代謝物產生的效果不佳，本實驗又將從 Neosartorya fischeri以分離出的二次代謝物作為抗菌試驗，發現在綠膿桿菌、沙門氏桿菌與大腸桿菌並無任何效果。關鍵詞：二次代謝產物， Neosartorya fischer， fusion PCR，Aspergillus nidulans(nkuAΔ) ，碳源，微量元素 Fungal secondary metabolites are an important source of bioactive compounds for agricultural and medical. A survey of the literature on 1,500 secondary metabolites isolated and characterized between 1993 and 2001 revealed that over half of these compounds have antibacterial, antifungal, or antitumor activity. In the past data, Neosartorya fischeri contains 16 sets of PKS and 18 sets of NRPS gene clusters to synthesize prediction of specific secondary metabolites. The present study, Neosartorya fischeri secondary metabolites related literature is very scarce, Therefore, from Neosartorya fischeri genome prediction has been shown, there are large number of secondary metabolites has not been found. This is probably because many secondary metabolite gene clusters are not expressed under normal laboratory culture conditions. In this study, the use of fusion PCR completed which NFIA_043670 fusion gene, and NFIA_043680, NFIA_043690 PyroA/WA3’cloning. In the experiment, with the change to a different carbon source to investigate the performance of the secondary metabolites, the use of glucose as a carbon source for the control group, monosaccharides of xylose, fructose and the disaccharide of sucrose and lactose as the experimental group, and the high performance liquid chromatography detection found that the area under the curve best performance, glucose carbon source is xylose carbon source, followed by fructose, sucrose, lactose manifested area under the curve worst, this experiment also added ammonium chloride as nitrogen sources found poor effect of secondary metabolites produced in this experiment in turn to separate out from Neosartorya fischerisecondary metabolites as antibacterial test, does not have any effect found in Pseudomonas aeruginosa , Salmonella and E. coli.
Relation:	校內五年後公開，校外永不公開，學年度：100，64頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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