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研究生:曾于銓
研究生(外文):Yu-Chuan Tseng
論文名稱:鑑定靈芝的新基因:以岩藻糖轉移酵素作為例子
論文名稱(外文):Identification of novel genes in Ganoderma lucidum: using fucosyltransferase as an example
指導教授:楊永正楊永正引用關係
指導教授(外文):Ueng-Cheng Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物醫學資訊研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:85
中文關鍵詞:靈芝岩藻糖轉移酵素新世代定序技術預測基因蛋白質模組
外文關鍵詞:ganoderma lucidumfucosyltransferasenext generation sequencingpredicted genedomain
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靈芝屬於真菌界擔子菌門,在亞洲地區是有名的傳統中藥材,過去研究中證實靈芝的多醣體萃取物對於癌症有治療的功效。另有研究指出多醣體末端以-1,2鍵結的岩藻糖為活性的關鍵,然而如何合成此多糖該合成酵素之基因目前尚未被發現。而先前自動化註解分析中有許多基因被註解為未知的功能,是因為在現有的資料庫中比對不到功能已知的基因。因此有需要將對靈芝的序列做進一步註解分析。
註解作業中我們採取序列守舊性的特性,利用pfam資料庫找出醣類相關的家族,透過排比資訊分析出可能與醣類鍵結的胺基酸,再建立具有保留性的新序列模組,以新的模組對靈芝序列進行比對搜尋,最後已序列比對的證據做初步驗證分析。
最後鑑定出靈芝的預測基因14270_g包含的特徵最符合,因此認為該基因可能為-1,2-岩藻糖轉移酵素。此方法針對醣類代謝酵素中具守舊性的序列,註解出靈芝中功能不明的醣類轉移酵素,不僅可應用在靈芝,未來對於其他的真菌物種也能進行相同的分析方式。
Ganoderma lucidum (GL), which is a basidiomycetous fungus, has been used to a traditional medicine in East Asia for thousands of years. Chemicals extracted from GL fruiting bodies are effective in the treatment of certain cancers in vitro as well as in vivo in model organisms. However, the biological pathways of synthesizing such chemicals remain unclear. Many enzymes can only be annotated with simple functional roles and their ligand specificities are usually unknown. In this study, we re‐annotate the protein domains in the GL genes that are likely to be implicated in such pathways. We retrieved protein families implicated in glycosylation from Pfam. The ligand specificity of each protein family was determined based on the heterogeneity of initial annotations of its seed sequences. For protein families that are less specific than others, each family was further refined to generate more specific domain models. New pHMMs so built were used to re‐annotate the GL genome. The predicted gene 14270_g is the most likely candidate for -1,2-fucosyltransferase. This method can improve the annotation of ligand specificity of glycosylation enzymes in GL. Moreover this method can be easily adapted to investigate the synthesis pathways of other secondary metabolites not only in GL but also in other fungal species. Our method can play a role in annotating the massive amount of genomic sequences being fast accumulated due to the recent advances of the next generation sequencing (NGS) technologies.
目錄
誌謝 i
中文摘要 ii
Abstract iii
目錄 iii
圖目錄 viii
表目錄 x
Chapter 1 緒論 1
1.1 引言:目前基因體分析的進展 1
1.1.1 真菌基因體的註解方法 2
1.1.2 已完成的真菌基因體計畫 3
1.2 靈芝的重要性 4
1.2.1 真菌界擔子菌門生長週期 4
1.2.2 靈芝孢子 5
1.2.3 靈芝菌絲體 5
1.2.4 靈芝子實體 6
1.2.5 靈芝多醣體功效 6
1.2.6 靈芝多醣體中的岩藻糖(Fucose) 6
1.3 靈芝的基因體定序計畫 8
1.4 靈芝的EST定序計畫 8
1.5 註解靈芝的困難點 9
1.6 本論文如何解決註解靈芝的困難 10
Chapter 2 材料與方法 11
2.1 資料來源 11
2.1.1 靈芝序列 11
2.1.2 真菌序列註解資料來源 12
2.2 分析工具 15
2.2.1 硬體設備 15
2.2.2 作業環境 15
2.2.3 軟體使用 16
2.2.4 自己撰寫的程式 21
2.3 註解用的流程 22
2.3.1 基因體註解 22
2.3.2 醣類結合位分析 23
2.3.3 分析醣類轉移酵素之候選基因與證據收集 26
2.4 靈芝網站與Ensembl網站 28
Chapter 3 分析策略 31
3.1 註解方法回顧 31
3.2 利用醣類結合位胺基酸的保留特性 32
3.3 利用EC編碼對應於化學反應 32
3.4 收集其他物種中某EC編碼分類的蛋白質序列 33
3.5 利用Pfam資訊尋找蛋白質序列保留性片段 33
3.6 測試保留性片段在某EC編碼分類蛋白質的一致性 34
3.7 建立HMM儲存不同物種蛋白質的保留性片段 34
3.8 搜尋DNA序列找出相似的區域 35
3.9 證據收集與分析候選基因的可能性 36
Chapter 4 結果 37
4.1 基因體註解 37
4.1.1 Uniprot資料庫註解 37
4.1.2 Pfam資料庫註解 37
4.2 酵素序列中與醣類結合的區域分析 38
4.2.1 整合化學結構、EC編碼、Domain資訊 38
4.2.2 Pfam Domain序列中的高保留性區域 40
4.2.3 資料庫蛋白質序列與高保留性片段的一致性 41
4.2.4 不同物種特定蛋白質序列建立MCR-HMM 43
4.2.5 測試MCR-HMM的正確性 45
4.2.6 比對PDB蛋白質的立體結構 47
4.2.7 靈芝Contigs上可能區域與預測基因的關聯 49
4.2.8 MCR-HMM比對其他真菌物種 52
4.3 候選基因的證據 53
4.3.1 蛋白質資料庫比對與Pfam domain註解 53
4.3.2 膜蛋白預測與分析 54
Chapter 5 討論 56
5.1 基因預測結果分析 56
5.2 基因序列上醣類結合位分析 57
5.3 基因註解結果分析 58
參考資料 61
附錄一、靈芝預測基因的註解數量分佈 65
附錄二、含五個以上醣類domain的預測基因 66
附錄三、靈芝EST: YMGLESTT51957比對資料庫的綜合結果 68
附錄四、靈芝預測基因g1575比對資料庫的綜合結果 69
附錄五、靈芝預測基因9520_g比對資料庫的綜合結果 72
附錄六、靈芝預測基因13462_g比對資料庫的綜合結果 75
附錄七、靈芝預測基因2542_g比對資料庫的綜合結果 78
附錄八、靈芝預測基因14270_g比對資料庫的綜合結果 81
附錄九、靈芝預測基因11899_g比對資料庫的綜合結果 84

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