表現序列標記（Expressed Sequence Tags；ESTs），是由生物體取得大量傳訊核糖核酸（messenger ribonucleic acid；mRNA）反轉錄之互補序列（complement DNA；cDNA）所建立的序列庫（cDNA library）中，隨機選取定序約400至600個鹼基（bases）的小片段序列，目前廣泛作為發掘新基因及分析基因表現的工具之一。本實驗室建立並分析10日齡莫三鼻克吳郭魚（Oreochromis mossambicus）腦部的EST序列庫（EST library），初步了解此齡莫三鼻克吳郭魚腦部的基因表現，以協助將來探究腦部兩性相（sexual dimorphism）關鍵時期，腦部神經發育、神經內分泌與性別分化的關係，及於腦部重要但未知之基因的發現。所建立之10日齡莫三鼻克吳郭魚腦部EST序列庫，總訂包括1,124個EST序列，可判讀者有1,092個，而去除判讀後過短（<200bp）及完全屬於載體的序列，可分析者有1,084個。可分析的吳郭魚腦部ESTs，經合併可產生108個疊連群（contigs）（包含261個ESTs）、20組相似群（包含40個ESTs），及783個非重複及相似的ESTs（singletons），這些ESTs以序列比對程式BLAST（Basic Local Alignment Search Tool）中之BLASTx（經轉譯之核酸-蛋白質比對）和BLASTn（核酸-核酸比對）分別依序與美國國家衛生研究院的國家生物技術資訊中心（NCBI；National Center of Biotechnology Information）之非重複蛋白質資料庫、GenBank中非重複核酸資料庫及EST資料庫dbEST比對後，發現結果如下：疊連群中分別有57個（含146個吳郭魚腦部ESTs）及16個（含37個吳郭魚腦部ESTs）與非重複之蛋白質資料庫及核酸資料庫中序列高度相關，其餘35個（含78個吳郭魚腦部ESTs）屬未知序列；相似群中分別有九組、一組及二組與非重複之蛋白質資料庫、核酸資料庫及dbEST中序列高度相關，其餘八組屬未知序列（每組各含兩個吳郭魚腦部ESTs）；而非重複及相似的ESTs中分別有309個、52個及47個與非重複之蛋白質資料庫、核酸資料庫及dbEST中序列高度相關，屬於未知序列者則有375個。整體來說，此一吳郭魚EST序列庫中，ESTs所與高度相關的蛋白質序列，以具有鍵結及傳輸功能者為最多。近幾年來，許多學者藉由ESTs的收集及EST序列庫的建立，為物種演化、個體基因組多型性與某一特定藥物影響的關係、一基因是否具其他同功異型物等相關問題提供了合理的解釋，相信此次建立的10日齡莫三鼻克吳郭魚腦部EST序列庫，有助於提升本實驗室對於所研究之方向中問題的解決能力。

The strategy of producing expressed sequence tags (ESTs) is greatly used for gene mining and analysis of gene expression. An EST is a partial and single-pass sequence generated from a complementary DNA (cDNA) library by random selection, and is typically about 400 to 600 bases. Here, we constructed and analyzed a brain EST library from 10-day-old (10-day posthatching) tilapia, Oreochromis mossambicus, to assist in investigating the relation between brain neural development and neuroendocrine and brain sexual differentiation during the critical period of sexual dimorphism by understanding brain gene expression, and, furthermore, to find genes that are important in brain but are unknown nowadays. A total 1,124 ESTs were in the 10-day-old tilapia brain EST library, and 1,092 ones were readable, and after discarding one that is too short (<200bp) or is totally vector sequence from the readable ESTs, 1,084 ESTs were assembled and then analyzed. 108 contigs from 261 ESTs, 20 kinds of similar sequences from 40 ESTs, and 783 singletons from the 1,084 ESTs were found, and after aligning in turn with the non-redundant (nr) protein database, nr nucleotide database and EST database dbEST supported by the National Center of Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) programs BLASTx (translated nucleotide-protein alignment) and BLASTn (nucleotide-nucleotide alignment) respectively, the results are as follows: 57 and 16 contigs including 146 and 37 ESTs matched with sequences in the nr protein and nr nucleotide databases respectively, and the rest 35 contigs including 78 ESTs were novel sequences; nine, one and two kinds of the similar sequences including 18, two and four ESTs matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest eight kinds of the similar sequences including 16 ESTs were novel sequences; 309, 52 and 47 singletons matched with sequences in the nr protein, nr nucleotide and dbEST databases respectively, and the rest 375 singletons were novel sequences. As a whole, the most functions of proteins that the contigs, similar kinds of sequences, and singletons from the 10-day-old tilapia brain EST library matched with were binding and transport activity. Recently, many researchers provide reasonable explanations for evolution, relation between genomic polymorphism and drug effects, and isoforms presentation of known proteins by collecting ESTs from open EST databases or from EST libraries they constructed, and we believe that this 10-day-old tilapia brain EST library can promote our ability to resolve questions about the topic we are researching in.

中文摘要
英文摘要
圖目錄
表目錄
論文本文
前言
實驗材料與方法
壹、經費來源
貳、吳郭魚腦mRNA的萃取及cDNA序列庫的建立
一、吳郭魚腦的取得
二、吳郭魚腦mRNA的萃取
三、cDNA序列庫的建立
參、吳郭魚腦部ESTs的分析
一、吳郭魚腦部ESTs校正
（一）去除殘留之載體序列與無法辨識的序列
（二）吳郭魚腦部ESTs的校正
（三）合併具重疊（overlapped）吳郭魚腦部ESTs及整合相似序列
二、吳郭魚腦部ESTs分析
（一）吳郭魚腦部ESTs與NCBI序列資料庫之序列相似度分析
（二）吳郭魚腦部ESTs註解
（三）與吳郭魚腦部ESTs有顯著相似之基因的分類
結果
壹、載體及adaptor序列污染之分析
貳、重複及相似ESTs之整合與序列比對結果
參、非重複及相似之ESTs的序列比對結果
一、BLASTx-nr結果分析
二、BLASTn-nr結果分析
三、BLASTn-est結果分析
肆、基因分類之分析統計
討論
參考文獻
圖
表

Adams, M.D., Dubnick, M., Kerlavage, A.R., Moreno, R., Kelley, J.M., Utterback, T.R., Nagle, J.W., Fields, C., and Venter, J.C. (1992). Sequence identification of 2,375 human brain genes. Nature 355:632-634.
Adams, M.D., Kelley, J.M., Gocayne, J.D. et al. (1991). Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252:1651-1656.
Areshchenkova, T. and Ganal, M.W. (2002). Comparative analysis of polymorphism and chromosomal location of tomato microsatellite markers isolated from different sources. Theor. Appl. Genet. 104:229–235.
Bentley, D.R. (2000). The Human Genome Project—an overview. Med. Res. Rev. 20(3):189–196.
Campos-Ramos, R., Harvey, S.C., Masabanda, J.S., Carrasco, L.A.P., Griffin, D.K., McAndrew, B.J., Bromage, N.R., and Penman1, D.J. (2001). Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis. Genetica 111:143-153.
Cao, D., Kocabas, A., Ju, Z., Karsi, A., Li, P., Patterson, A., and Liu, Z. (2001). Transcriptome of channel catfish (Ictalurus punctatus): initial analysis of genes and expression profiles of the head kidney. Animal Genetics 32:169-188.
Hillier, L., Lennon, G., Becker, M. et al. (1996). Generation and analysis of 280,000 human expressed sequence tags. Genome Res. 6:807-828.
Ju, Z., Karsi, A., Kocabas, A., Patterson, A., Li, P., Cao, D., Dunham, R., and Liu, Z. (2000). Transcriptome analysis of channel catfish (Ictalurus punctatus): genes and expression profile from the brain. Gene 261:373-382.
Kocher, T.D., Lee, W.J., Sobolewska, H., Penman, D., and McAndrew, B. (1998). A genetic linkage map of a cichlid fish, the tilapia (Oreochromis niloticus). Genetics 148:1225–1232.
Kwon, J.Y., McAndrew, B.J., and Penman, D.J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Mol. Reprod. Dev. 59:359-370.
Oliveira, C., Chew, J.S., Porto-Foresti, F., Dobson, M.J., Wright, J.M. (1999). A LINE2 repetitive DNA sequence from the cichlid fish, Oreochromis niloticus: sequence analysis and chromosomal distribution. Chromosoma 108(7):457-468.
Pontius, J.U., Wagner, L., and Schuler, G.D. (2002). Chapter 20: UniGene: a unified view of the transcriptome. In The NCBI Handbook, sited on http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=handbook.TOC&depth=2 (Copyright by NCBI website, Bethesda, MD, USA)
Sharp, F.R., Bernaudin, M., Bartels, M., and Wagner, K.R. (2001). Chapter 34: Glial expression of heat shock proteins (HSPs) and pxygen-regulated proteins (ORPs). In Progress in brain research: glial cell function, Vol. 132, B.C. Lopez and M. Nieto-Sampedro eds. (Amsterdam, New York, Elsevier Press), p427-429.
Sorek, R. and Safer, H.M. (2003). A novel algorithm for computational identification of contaminated EST databases. Nucleic Acids Res. 31(3):1067-1074.
Tsai, C.L., Chang, S.L., Wang, L.H., and Chao, T.Y. (2003). Temperature influence the ontogenetic expression of aromatase and oestrogen receptor mRNA in the developing tilapia (Oreochromis mossambicus) brain. J. Neuroendocrinol. 15(1):97-102.
Tsai, C.L., Wang, L.H., Chang, C.F., and Kao, C.C. (2000). Effects of gonadal steroids on brain serotonergic and aromatase activity during the critical period of sexual differentiation in tilapia, Oreochromis mossambicus. J. Neuroendocrinol. 12(9):894-898.
Tsai, C.L. and Wang, L.S. (1999). Effects of gonadal steroids on the serotonin synthesis and metabolism in the early developing tilapia brain. Neurosci. Lett. 264(1-3):45-48.
Tsai, C.L., Wang, L.S., and Fang, L.S. (2001). Estrogen and para-chlorophenylalanine downregulate the expression of brain aromatase and estrogen receptor-α mRNA during the critical period of feminization in tilapia (Oreochromis mossambicus). Neuroendocrinology 74(5):325-334.
Wolfsberg, T.G. and Landsman, D. (1997). A comparison of expressed sequence tags (ESTs) to human genomic sequences. Nucleic Acids Research 25(8):1626-1632.
Wolfsberg, T.G. and Landsman, D. (2001). Chapter 12: Expressed Sequence Tags (ESTs). In Bioinformatics: a practical guide to the analysis of gene and proteins, 2nd Edition, A.D. Baxevanis and B.F.F. Ouellette eds. (A John Wiley & Sons, Inc. Press), p283-302.