臺灣博碩士論文加值系統

中文摘要
水稻(Oryza sativa L.)是我國最主要之糧食作物，重要蟲害為褐飛蝨(brown planthopper, BPH ; Nilaparvata lugens (Stal.))，而病害以稻熱病(rice blast disease)為主。表現序列標幟(Expressed Sequence Tag, EST)是cDNA序列片段，直接研究EST可獲取基因表現訊息。為瞭解造成水稻褐飛蝨和稻熱病抗性基因的功能機制，本研究首先擷取NCBI之dbEST資料庫中受褐飛蝨誘導之不同水稻抗、感性品種的EST資料(共188筆)，進行功能註解之基因表現差異比較，並建立了一套水稻EST功能註解的分析流程：將水稻EST資料經CAP3軟體聚集和接合後所獲得的較高品質和去除多餘性的單一序列(unique sequences)，去比對TIGR(The Institute for Gene Research)之TC(tentative consensus sequence)資料庫以進行功能註解，再藉由TC搜尋結果連結到GO(Gene Ontology)與MIPS(Munich Information Center for Protein Sequences)分類系統進行功能分類，最後進行基因表現量之統計。然後以此分析流程為基礎，進一步在Linux操作平台上構築本端資料庫及利用Perl語言撰寫程式，建立了EST功能註解自動化分析系統，對dbEST資料庫中不同水稻品種、植株部位及發育時期的受稻熱病菌誘導之巨量水稻EST資料(共84,705筆)進行功能註解，以瞭解水稻於不同品種、不同發生部位或不同發育時期受稻熱病菌誘導的基因表現差異特性。分析結果顯示：(1)受褐飛蝨誘導之水稻抗、感品種在GO功能分類上，皆以生物過程(biological process)的功能表現為主；而在更細分的MIPS功能分類上，抗性品種在生物過程中的代謝(metabolism)相關基因的表現量較感性品種為高，同時抗性品種啟動了感性品種所沒有的細胞援救、防禦和毒性(cell rescue、defense and virulence)與轉錄(transcription)與細胞生長/型態發生(cell growth / morphogenesis)此三種功能。(2)受稻熱病菌誘導的水稻EST序列若混合了不同品種及不同部位組織，與使用單一品種及固定部位組織在基因表現上有明顯差異；野生稻受稻熱病菌誘導之EST的代謝(metabolism)功能之表現量高於秈稻和稉稻。這些發現，有利於水稻對病蟲害之基因防禦機制之研究。

Abstract

Rice (Oryza sativa L.) is the most important crop in the world. Brown planthopper is the major pest so is the rice blast disease the most serious problem. Expressed sequence tag (EST) is the fragment of cDNA sequence. Therefore, direct studies of EST is helpful to obtain the gene expression information. In order to know the functional mechanisms of the resistant gene which cause the brown planthopper and rice blast, this study firstly accessed rice EST sequences induced by brown planthopper from NCBI-dbEST database (with the total amounts as 188), and compared the gene expression difference between the resistant and sensitive varieties of rice. An analytic procedure of rice EST functional annotation was established as: Firstly, using unique sequences of high quality from removing redundancy after clustering and assembling by CAP3 software to homology search TC(tentative consensus sequence) database of TIGR (The Institute of Genomic Research), then linking to the functional catalogs of GO (Gene Ontology) and MIPS (Munich Information Center for Protein Sequences) from tentative annotation of the selected TC sequences, and finally calculating the statistics of gene functional representation. Based on this procedure, an automated system of functional annotation was developed. All programs of this system were written in perl language under local Linux platform. The automated system was implemented in the huge amount of rice ESTs induced by M. grisea (with the total amounts as 84,705) to investigate the gene expression differences among different varieties, tissues and developmental stages of rice. The results showed that (1) based on GO functional category, biological process was the dominant gene function of rice ESTs induced by brown planthopper. According to minute classification of MIPS functional category system, the genes of resistant rice variety expressed richer metabolism function than that of sensitive rice variety. The resistant rice variety switched on the functions related to cell rescue/defense and virulence, transcription, and cell growth/morphogenesis, but sensitive variety did not. (2) When rice ESTs induced by M. grisea were mixtures of sequences with different varieties, tissues or developmental stages, it appeared diversity of gene expression from those of rice ESTs from single source. The genes of wild rice expressed richer metabolism function than those of Indian rice and Japanese rice. The findings of this study will help clarify the mechanisms of rice genes resistant to disease and pest.

目錄
中文摘要…………………………………………………………………….…Ⅰ
英文摘要…………………………………………………………………….…Ⅲ
誌謝………………………………………………………………………….…Ⅴ
表目錄……………………………………………………………………….…Ⅷ
圖目錄……………………………………………………………………….…Ⅸ
壹、前言…………………………………………………………………………1
貳、前人研究……………………………………………………………………3
一、水稻重要病蟲害方面之研究
二、EST發展與應用之研究
三、禾穀類作物EST資料之功能註解研究
四、有關功能註解之相關網路資源之簡介
五、Perl、PHP程式語言與MySQL資料庫之簡介
參、材料與方法……………………………………………………………….16
一、試驗材料
二、EST的聚集與接合
三、EST功能註解分析流程之建立
四、EST功能註解自動化分析系統之建立
五、基因庫與功能關係之探討
肆、結果……………………………………………………………………….23
一、EST的聚集及接合
二、受褐飛蝨誘導之水稻EST的功能註解
三、水稻EST功能註解自動化分析系統之建立及效益評估
四、受稻熱病菌誘導之水稻EST的功能註解
伍、討論…………………………………………………………………….…31
陸、參考文獻……………………………………………………………….…35
柒、表……………………………………………………………………….…42
捌、圖………………………………………………………………………….53
附錄、受褐飛蝨誘導之水稻EST單一序列經TGI資料庫進行相似性比對的結果………………………………………………………………….…72
簡歷………………………………………………………………………….…81

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