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研究生:黃雅芳
研究生(外文):Ya-Fang Huang
論文名稱:利用D2LSU、ITSrDNA序列分析樟芝與薄孔菌屬的親緣關係及簡易快速之鑑定技術開發
論文名稱(外文):Phylogenetic Analysis of Antrodia camphorata and Antrodia species based on D2 LSU and ITS rDNA Sequences, and develops simply and rapid identification technique
指導教授:邱華賢邱華賢引用關係
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:生物技術系碩士班
學門:生命科學學門
學類:生物科技學類
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:47
中文關鍵詞:ITSITS-PCR RFLP樟芝薄孔菌屬rDNA親緣關係樹狀圖D2 LSU
外文關鍵詞:phylogeneticribosomal DNAAntrodiaAntrodia camphorateITS-PCR RFLPITSD2 LSU
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樟芝為台灣特有的真菌,具極高之宿主專一性,只生長在台灣保育樹種牛樟樹(Cinnamomum kanehirai)的中空樹幹中。 本研究目的是運用分子生物鑑定技術中的D2 LSU 與ITS rDNA的序列分析樟芝與薄孔菌屬的親源關係。 本研究發現樟芝的D2 LSU rDNA序列呈現100%序列相似度,在D2區域中序列長度均為302 bp。 在親緣關係樹狀分析圖顯示,薄孔菌屬中同一菌種的D2 LSU rDNA在親緣關係演化上為同一群(group),且具有98% 以上的bootstrap值的信賴度支持演化分析圖,D2 LSU rDNA序列適合種級以上分類研究;在薄孔菌屬中ITS rDNA序列總長度範圍介於543 bp-610 bp、序列相似度在99.3-65.6%之間。 在ITS的親緣關係樹狀分析圖中,同菌種的ITS rDNA序列,在親緣關係演化上雖為同一群,但不同菌株可經由演化距離清楚地區分。 依據本研究樟芝序列的分析結果得知,ITS比D2 LSU rDNA序列的演化速度快,常被用來研究同屬中種間的研究。 在親緣關係演化結果呈現,樟芝與薄孔菌屬在D2 LSU與ITS rDNA的演化上均在同一族群,本研究依據D2 LSU與ITS rDNA的親緣關係樹狀分析結果,建議將樟芝應歸屬於薄孔菌屬。 為尋求更簡易且快速的樟芝菌種篩檢技術,我們運用ITS-PCR RFLP的方法,發現限制酶Bbv I的切位點,可以輕易的區別樟芝與其他薄孔菌屬之間;利用樟芝菌種的SNP運用於限制酶Sap I可作為樟芝種內菌株的判別,本研究利用ITS-PCR RFLP分子鑑定方法成功建立簡易快速的篩檢樟芝技術,提供日後真菌鑑定技術發展的基礎。
Medicinal fungus Antrodia camphorata is one of the most valued mushroom because it grows slowly only on the Cinnamomum kanehirai. The goal of this study was to test D2 LSU and ITS rDNA for identifying A. camphorata and related taxa. The molecular approach typically use to identify fumgi is usually based upon the sequence analysis of the incorporated ribosomal DNA (rDNA). This approach is based upon a concept of highly conserved DNA sequences arising as a consequence of evolution as also highly variable sequences arising amongst species, and even, on occasion, within species. Divergence in D2 domain of the LSU rDNA sequence is generally sufficient to resolve individual species. According to the sequence data from D2 region of LSU rDNA, phylogenetic analysis of this study allows to infer most combination of strains in the same species exhibited a level of bootstrap value of 98% or more. A method to identify Antrodia fungi at the strains and species level, was developed herein, using an internal transcribed spacer (ITS) region. ITS amplicons from Antrodia species ranged in size from 543 to 610bp. ITS size of A. camphorata strains ranged from 592 to 596bp. The ITS sequence similarities of various A. camphorata strains examined ranged from 98.4% to 99.3%. Eight other Antrodia species demonstrated less than 71% similarity when compared with the A. camphorata ITS sequence. Polymorphisms arising within ITS1-5.8S-ITS2 region can provide practical markers to establish a foundation for the further expansion of ITS sequence database of medically important fungi. The inter-strain comparison of ITS-PCR RFLP sequences of Antrodia strains performed in this work revealed some variability as regards DNA fragment length. From biotechnological point of view, ITS-PCR RFLP would appear to feature a rather substantial potential application for the purposes of product ID and quality control of A. camphorata strains for the commercial production for sale of this fungus.
第一章 緒論
第二章 文獻探討
第一節 樟芝第二節 薄孔菌屬
第三節 樟芝命名之演進
第四節 真菌的rDNA序列
第五節 真菌的ITS rDNA
第六節 ITS-PCR限制酶片段長度多型性
第三章 材料與方法
一、 實驗菌株
二、 實驗菌種培養
三、 樟芝之菌種保存
四、 DNA萃取
五、 D2 LSU rDNA PCR
六、 ITS rDNA PCR
七、 PCR產物的純化
八、 螢光劑反應
九、 酒精沉澱
十、 DNA定序
十一、 基因序列之比對分析方法
十二、 親緣關係樹狀圖分析方法
十三、 限制酶之選擇
十四、 ITS PCR與電泳分析
十五、 ITS-PCR RFLP
第四章 結果與討論
一、 樟芝與其他菌屬之D2 LSU rDNA序列結果
二、 樟芝與薄孔菌屬D2 LSU rDNA序列比對結果
三、 樟芝與薄孔菌屬D2 LSU rDNA的演化樹分析
四、 樟芝ITS rDNA序列分析結果
五、 樟芝與薄孔菌屬ITS rDNA序列比對結果
六、 樟芝與薄孔菌屬ITS rDNA的演化樹分析
七、 ITS rDNA的電泳分析結果
第五章 結論與建議
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