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研究生:林奕承
研究生(外文):Yi-Chen Lin
論文名稱:水稻白葉枯病抗性基因多白胺酸重複(LRR)的比較分析
論文名稱(外文):Comparative Analysis of Leucine-Rich Repeat (LRR) in Rice Bacterial Blight Resistance Genes
指導教授:呂秀英呂秀英引用關係
指導教授(外文):Hsiu-Ying Lu
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:98
中文關鍵詞:同義密碼子相對使用頻率同義密碼子相對使用度抗性基因一致序列多白胺酸重複序列對應分析水稻白葉枯病高頻密碼子最小顯著性測驗水稻分子生物百科資料庫變方分析蛋白質家族資料庫蛋白質結構資料庫
外文關鍵詞:resistant geneRFSCRSCUrice bacterial blightCorrespondance analysisLRRconsensus sequenceHFLSDKOMEPDBPfamANOVA
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水稻白葉枯病 (rice bacterial blight) 是水稻嚴重的世界性病害,故水稻白葉枯病抗病基因(Xa)之研究相當重要。目前已知多白胺酸重複(leucine-rich repeat, LRR)為生物體抗病基因特有的序列,而且在水稻的Xa1、Xa21及Xa26基因中也發現LRR存在,因此本研究自NCBI及KOME公用資料庫內擷取出此三個Xa基因的已知CDS與cDNA序列,再將其轉譯的蛋白質序列利用Pfam資料庫尋找出各LRR片段,進而利用生物資訊與統計方法,進行不同Xa基因LRR序列的比較分析。經由分析結果顯示:Xa1基因的LRR在蛋白質序列上的分布較疏鬆分散,而Xa21及Xa26基因的LRR之分布則緊密集中。三個Xa基因之LRR的胺基酸組成皆以親水性胺基酸為最多且其中以白胺酸 (leucine, Leu) 出現頻率最高,其次為不帶電荷之絲胺酸 (serine, Ser) ,另有7種胺基酸之使用在不同Xa基因間呈顯著差異。三個Xa基因之密碼子偏好性各有不同,以Leu而言,Xa1偏好CUC,而Xa21偏好CUG,Xa26則無任何偏好;以Ser而言,Xa1和Xa26皆偏好UCU,而Xa21則無偏好。最後將三個Xa基因LRR序列經ClustalW多序列排比結果,得到Xa基因LRR之β-sheet一級結構序列的模組樣式(motif pattern)。此外,為能快速且準確完成各基因之胺基酸偏好性、密碼子偏好性與高頻密碼子確定等比較分析,利用SAS軟體之程序語言,撰寫程式以建立一套自動化分析系統,大大提高了工作效率。本研究之結果發現與自動化分析系統,有助於Xa基因之LRR序列結構之相關研究。
Rice bacterial blight is a devastating rice disease throughout the world. The study on Xa, a bacterial blight resistance gene, is therefore of great significance. As a leucine-rich repeat (LRR), known as a sequence motif specific to resistance genes, is also found in some rice bacterial blight resistance genes such as Xa1, Xa21 and Xa26, this study retrieves the given CDS and cDNA sequences of these three Xa genes from the NCBI and KOME databases and, by using the Pfam database, identifies each LRR fragment from the translated protein sequences. With bioinformatics and statistical approaches, we further conduct comparative analyses of LRR sequences among diverse Xa genes. According to the analysis results, the distribution of Xa1 LRRs is more dispersed on protein sequences, in contrast with that of Xa21 and Xa26 LRRs, which is more intensive. The LRRs of these three Xa genes mainly consist of hydrophilic amino acids, in which leucine (Leu) shows the highest frequency of presence, followed by neutral serine (Ser). In addition, there are significant differences in the use of Xa genes among seven amino acids. The three Xa genes are characterized by unique codon bias. In terms of Leu, for example, Xa1 has a bias in favor of CUC and Xa21 in favor of CUG, while Xa26 has none. In terms of Ser, both Xa1 and Xa26 have a bias in favor of UCU, while Xa21 has none. At last, a motif pattern of β-sheet primary structure sequence is derived from the three Xa LRR sequences through the ClustalW multiple sequence alignment. Besides, to complete the comparative analyses of the preference of amino acids, codon biases and high-frequency codon rapidly and accurately, we use the SAS software language to program an automated analysis system that dramatically improves the operating efficiency. The findings and the automated analysis system of the study are helpful for relevant research on Xa LRR sequences and structures.
中文摘要.................................................I
英文摘要.................................................II
誌謝.....................................................III
目錄.....................................................IV
圖目錄.......................................................VIII
表目錄...................................................IX

第一章、 前言............................................1

第二章、 前人研究........................................4
2.1 水稻白葉枯病抗性基因與抗病基因之研究.................4
2.1.1 Xa1基因...........................................5
2.1.2 Xa21基因..........................................6
2.1.3 Xa26基因..........................................6
2.1.4 Xa27基因..........................................7
2.1.5 Xa13基因..........................................7
2.1.6隱性基因xa5及xa13..................................8
2.2 R基因的表現蛋白質抗病機制與分類......................9
2.3 LRR序列的特性........................................10
2.3.1 LRR結構域.........................................10
2.3.2 NBS-LRR表現蛋白結構...............................11
2.4 蛋白質序列的胺基酸偏好性分析.........................12
2.5胺基酸同義密碼子偏好性分析............................13
2.5.1密碼子的簡併性.....................................13
2.5.2基因表達程度與天擇.................................15
2.5.3 mRNA的轉譯速率....................................15
2.6 高頻密碼子決定.......................................17
2.6.1 最優密碼子........................................17
2.6.2 同義密碼子相對使用頻率............................17

第三章、 材料與方法......................................19
3.1 Xa基因蛋白質序列的蒐集與整理.........................19
3.2 Xa基因蛋白質之LRR序列片段的搜尋及其分布探討..........20
3.3 Xa基因蛋白質之LRR序列3D結構的功能註解................21
3.4 Xa基因蛋白質之LRR的胺基酸偏好性分析..................22
3.4.1 胺基酸組成........................................22
3.4.2 胺基酸屬性含量....................................23
3.4.3 GC胺基酸豐富度....................................24
3.5 Xa基因蛋白質LRR之密碼子偏好性分析....................25
3.6 Xa基因蛋白質LRR之高頻密碼子確定......................26
3.7 Xa基因蛋白質LRR之一致序列尋找........................26
3.8 SAS自動化分析系統之撰寫..............................27

第四章、 結果與討論......................................29
4.1 Xa基因蛋白質序列蒐集的結果...........................29
4.1.1 NCBI資料庫蒐集結果................................29
4.1.2 KOME資料庫蒐集結果................................30
4.2 Xa基因蛋白質之LRR序列片段及其分布結果................31
4.2.1 Pfam資料庫搜尋結果................................31
4.2.2 LRR蛋白質序列分布結果.............................32
4.3 Xa基因蛋白質之LRR序列的3D結構功能註解結果............32
4.4 Xa基因蛋白質之LRR的胺基酸偏好性分析結果..............33
4.4.1胺基酸組成.........................................33
4.4.2胺基酸屬性含量.....................................35
4.4.3 GC胺基酸豐富度....................................36
4.5 Xa基因蛋白質LRR之密碼子偏好性分析結果................36
4.6 Xa基因蛋白質之LRR的高頻密碼子確定結果................38
4.7 Xa基因蛋白質LRR之一致序列尋找結果....................39
4.8 SAS自動化分析系統之建立..............................41

第五章、 結論............................................43

參考文獻.................................................47

圖目錄...................................................56
圖1.本研究分析之架構流程圖...............................56
圖2.Xa基因蛋白質序列之LRR片段對應其cDNA或CDS上位置的示意圖.......................................................57
圖3.Xa蛋白質序列之LRR位置分布圖..........................58
圖4.PDB資料庫搜尋出LRR相似序列之3D蛋白質結構的輸出結果...59
圖5.Xa基因之LRR的胺基酸組成差異的雷達圖..................60
圖6.Xa基因之LRR胺基酸組成的對應分析雙標圖................61
圖7.Xa基因之LRR的胺基酸屬性含量差異的雷達圖..............62
圖8.Xa基因之LRR同義密碼子相對使用度( RSCU )的對應分析雙標圖.......................................................63
圖9. 本研究所研發之SAS自動化分析系統的分析策略流程圖.....64

表目錄...................................................65
表1.已知序列之水稻白葉枯病抗性基因相關資訊...............65
表2.水稻白葉枯病抗性基因cDNA序列相關資訊.................66
表3.Xa基因蛋白質序列於Pfam資料庫搜尋到的LRR數量及位置....67
表4.Xa基因各來源序列之LRR的胺基酸組成(%).................69
表5.不同Xa基因之LRR的胺基酸組成平均值比較................70
表6.不同Xa基因之LRR的胺基酸組成變異性(CV,%)比較..........71
表7.Xa基因各來源序列之LRR的胺基酸屬性含量(%).............72
表8.不同Xa基因之LRR的胺基酸屬性含量比較..................73
表9.Xa基因各來源序列之LRR的GC-rich和GC-poor胺基酸含量(%)及其間之比例...............................................74
表10.不同Xa基因之LRR的GC-rich和GC-poor胺基酸含量(%)及其間之比例比較.................................................75
表11.Xa基因LRR序列對應在已知CDS或cDNA序列上的位置........76
表12.Xa基因各來源序列之LRR的同義密碼子偏好性相對使用度(RSCU)...................................................78
表13.不同Xa基因之LRR的同義密碼子偏好性相對使用度(RSCU)平均值比較...................................................80
表14.Xa基因之LRR的同義密碼子偏好性相對使用度(RSCU) 變異性(CV,%)的比較.............................................81
表15.Xa基因各來源序列之LRR的同義密碼子偏好性相對使用頻度(RFSC,%).................................................82
表16.根據同義密碼子偏好性相對使用頻度(RFSC)所篩選之Xa基因的高頻密碼子...............................................84
表17.利用ClustalW所找出之Xa基因之LRR的一致序列...........86

附錄....................................................87
附錄1.水稻白葉枯病病徵圖................................87
附錄2.LRR結構之帶狀圖...................................88
附錄3.本研究所使用的各資料庫之網址及其功能概述..........89
附錄4.Pfam資料庫搜尋LRR序列片段輸出結果圖例.............90
附錄5.胺基酸屬性分類表..................................91
附錄6.各種胺基酸之同義密碼子表..........................92
附錄7.本研究所研發之SAS自動化分析系統之程式碼...........93

個人簡歷................................................98
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