水稻之基因組已完全解序，因此，在後基因體時代，研究水稻的基因及其功能之方法以T-DNA插入法為主流，T-DNA插入突變以T-DNA為tag，進一步由兩翼序列之獲得以瞭解可能的基因與功能之關連。對於逆向遺傳學的研究來說，獲得 T-DNA 插入突變點之兩翼序列(Flanking sequence , FS) 是不可或缺的，為水稻功能性基因體研究創造出非常有價值之資訊。我們可以藉由T-DNA之兩翼序列，來定位T-DNA序列在水稻基因體中的插入位置，再尋找附近可能影響突變的基因，提供給生物學家作進一步的性狀與基因之比較驗證。

由於，在比對T-DNA插入之兩翼序列的過程中，需要面對實驗室中PCR實驗的許多不確定因素，故往往需要反覆人工操作網站上提供的BLAST比對水稻基因體與質體序列資料庫，才能確認此為成功的FS片段，相當耗費時間與人力資源。

本研究使用BLAST結合perl語言撰寫而成『水稻T-DNA插入突變體兩翼序列自動化比對系統』，分別將T-DNA序列、質體序列及水稻基因體序列建立資料庫，取代網站的BLAST功能，設定期望值參數，並且能夠自動化針對序列進行篩選，讓使用者不需具備相關專業知識，也可以完成所有比對流程；此系統之建立達到加速序列比對過程，避免人為錯誤，且可準確獲得T-DNA在Rice genomic之位置。作為水稻FS實驗室後端的生物資訊處理與分析。由於快速的後端處理，輔助提升實驗的效率與成功率，也確保FS之正確性及機密性。目前經本系統比對之序列，已有超過3000條提供至中央研究院。

After the rice genome sequencing project is finished, functional genomic in rice by T-DNA insertional mutagenesis become the main stream of post genome era. In regard of reverse genetic research, obtaining T-DNA Flanking Sequence(FS) become the ultimate goal in rice functional genomic. By detecting the FS of T-DNA insertion location in rice genome, these flanking sequence can be utilized by the biologist to compare and identify the relationship between genotype and phenotype.

The alignment result of flanking sequence is time consuming and laborious in indentify of successful flanking sequence, moreover, BLAST is too often to repeat for aligning rice genome and plasmid database.

In this thesis『The establishment of T-DNA insert mutant flanking sequence automation BLAST system in rice』, we code PERL programming language by combining with BLAST, and to establish database of T-DNA sequence、plasmid sequence and rice genome sequence; by using this system can speed up the process and be analyze of bioinformation. By using this system, it can work faster and accurate to obtain the correct T-DNA location in rice genomic. In addition, this system can assure the accuracy and the confidentiality of the flanking sequence. Now, over 3000 sequence are already uploaded to Academia Sinica by this system.

目錄
1 緒論-1
1.1研究背景-1
1.2研究動機與目的-3
1.3論文架構-4
2 相關研究-5
2.1 功能性基因體研究-5
2.1.1 IRGSP-5
2.1.2 T-DNA(transfer-DNA-7
2.1.3 TRIM-10
2.2 BLAST介紹-11
2.3 決策樹-14
3 材料與方法-15
3.1 研究材料-15
3.1.1 序列來源-15
3.1.2 軟體及硬體-16
3.2 研究方法-17
3.2.1 系統架構-17
3.2.2 參數取得-19
3.2.3 建置BLAST資料庫-24
3.2.4 資料擷取-26
4 實驗結果-30
4.1 實際測試-30
4.2 結果-35
5 討論36
5.1 自動化系統與人工方法比較-36
5.2 T－DNA插入染色體統計-37
5.3 序列篩選-38
5.4 波形圖辨識-40
5.5 實驗解序結果統計-43
6 結論-44
參考文獻-46
附錄-49

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