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研究生:徐鈺惠
研究生(外文):Hsu, Yu-Hui
論文名稱:阿拉伯芥轉譯通讀基因之預測與分析
論文名稱(外文):Prediction and analysis of translational termination readthrough in Arabidopsis
指導教授:林彩雲林彩雲引用關係
指導教授(外文):Lin, Tsai-Yun
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:74
中文關鍵詞:阿拉伯芥轉譯通讀
外文關鍵詞:Arabidopsisreadthrough
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終止密碼子的轉譯通讀會產生額外的羧基端胜肽,造成終止密碼轉譯通讀的mRNA訊號在植物中的研究較少。造成終止密碼的轉譯通讀現象的mRNA序列在各物種間可能有特徵上的共通點,這些共同特徵可以用來預測可能會發生終止密碼子轉譯通讀的基因。藉由黃鎮剛教授和黃存操所編寫的電腦程式,我們分析阿拉伯芥基因的終止密碼子前後的序列,篩選出547個可能會發生終止密碼子轉譯通讀的基因,並利用雙報導基因NAN-GUS系統短暫表現在阿拉伯芥的原生質體檢測轉譯通讀的效率。由於菸草鑲嵌病毒有轉譯通讀現象的UAG終止密碼子在阿拉伯芥細胞有顯著的轉譯通讀效率,我們比較這547個基因的終止密碼子前後各12個鹼基(不包含終止密碼子),篩選出10個與菸草鑲嵌病毒UAG終止密碼子前後序列有50%以上相似度的阿拉伯芥基因。以NAN-GUS系統檢測,發現其中7個有轉譯通讀現象,但在阿拉伯芥中未發現實際存在的mRNA。由結果得知,若終止密碼子前後序列與菸草鑲嵌病毒UAG終止密碼子前後序列相似,有發生轉譯通讀現象的機會。我們重新設定準則分析阿拉伯芥基因,篩選出9個可能有轉譯通讀現象的基因,這些基因與菸草鑲嵌病毒UAG終止密碼子前後12個鹼基序列有50%相似(不包含終止密碼子),只有一種基因模組,在足夠長(120個鹼基)的三端非轉譯區域外另有一終止密碼子,且經由轉譯通讀,在兩終止密碼子之間可能轉譯出額外的功能性羧基端胜肽。這些實驗結果提供我們分析阿拉伯芥中可能會發生終止密碼子轉譯通讀基因的鑑定訊息。
Termination codon readthrough is utilized for extension of C-terminal protein domain. Signals in mRNA that promote translation readthrough have not been characterized in plant. The surrounding context of readthrough stop codon may contain conserved sequence feature and can be utilized for prediction of potential readthrough candidates. Using computational method designed by Dr. JK Huang and TT Huang, we analyzed the stop codon context in Arabidopsis genes and identified 547 potential candidates. The desired sequence was inserted in NAN-GUS dual reporter system and transiently expressed in Arabidopsis protoplasts to analyze readthrough efficiency. The tobacco mosaic virus (TMV) leaky UAG stop codon showed significant readthrough in Arabidopsis protoplasts. Thus the sequence context containing 12 nucleotides flanking both sides of the first stop codon of each candidate was compared to the context of the TMV leaky UAG stop codon. Ten candidates were selected with > 50% sequence identity, and readthrough activity was detected in seven sequences. We further analyzed Arabidopsis database using 5 criteria including > 50% identity to the context of TMV leaky UAG stop codon, containing a single gene model, having an in frame stop codon in 3' UTR more than 120 nucleotides, and extending a functional domain, and 9 candidates were extracted. This study provides information for identifying potential readthrough genes in Arabidopsis.
摘要 i
Abstract ii
謝誌 iii
Table of Content iv
List of Tables vii
List of Figures viii
List of Appendixes x
Abbreviations 1
Introduction 3
Materials and Methods 11
1. Plant material 11
2. Basic pipeline for extraction of readthrough candidate genes 11
3. Extraction of readthrough candidate genes using five criteria 11
4. Construction of dual reporter systems 13
■ Preparation of vector for cloning 13
■ Preparation of inserts for cloning 14
■ Completed constructs 15
5. Transient expression in Arabidopsis protoplasts 16
■ Preparation of Arabidopsis protoplasts 17
■ PEG-mediated protoplast transformation 18
■ Quantification of β-glucuronidase (GUS) activity 19
■ Quantification of bacterial sialidase (NAN) activity 19
6. Reverse transcription PCR (RT-PCR) for candidate genes 20
7. General procedures for construct preparation and bacterial transformation 20
■ DNA ligation 20
■ Competent cell preparation 20
■ Bacterial transformation 21
8. Plasmid DNA amplification and purification of DNA fragment 21
■ Mini-preparation of plasmid DNA 21
■ Large-scale plasmid DNA preparation 22
■ Purification of DNA fragment 23
9. Transient assay in Arabidopsis leaves using particle bombardment 23
10. Histochemical staining of GUS activity 24
Results 25
1. Computational analysis of the readthrough candidates in Arabidopsis 25
2. Analysis of readthrough efficiency of At3g53970 25
3. The 547 candidate genes were filtered based on the similarity of stop codon context in comparison to TMV leaky UAG stop codon 28
4. Re-screening of the readthrough candidates in the Arabidopsis database 31
Discussion 32
1. The readthrough efficiency of translational termination can be affected by the surrounding context of stop codon 32
2. Translational termination readthrough may be affected by stress 34
3. The length of nucleotides flanking the stop codon may affect readthrough efficiency 35
4. Intervention of other factor that may promote readthrough efficiency 36
References 37
Tables 48
Figures 53
Appendixes 67


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