臺灣博碩士論文加值系統

白點症（White spot syndrome，WSS）是一個由病毒所引起，在養蝦產業上造成鉅量經驗損失的病症，而且不只發生在台灣，白點症是具全球衝擊性的。然而，目前有關於宿主與病毒之間分子層次的調控作用上的資料十分的有限。先前有關白點症的研究著重在某個特定的器官上進行探討，但是白點病毒感染蝦子的影響是系統全面性的，而非只有某個特定器官的病變而導致病因，因此，在這個研究上，我們採用了草蝦的蝦苗，利用表現序列標籤（expressed se-quence tag，EST）的技術，試圖以全面性的角度，分析白點病毒與宿主細胞之間的基因調控關係。我們從正常以及白點病毒感染的cDNA clones，採3’ 的定序方向所得到的ESTs，一共分析了13.969條高品質的ESTs。我們的分析過程，包括與公開資料庫比對已知功能的基因、分析經白點病毒感染過後表現量上升或下降的基因、以Gene Ontology（GO）和Kyoto Encyclopedia of Genes and Genomes (KEGG) 生物代謝路徑為基礎的功能性分類分析等。在我們的分析結果中，46%的ESTs比對到公開資料庫National Center of Biotech-nology Information (NCBI) non-redundant (nr) 上已知的基因，44%具有GO功能註解，31%具有KEGG生物代謝路徑的註解。我們的研究，針對了由草蝦蝦苗得到13,969條高品質的ESTs進行全面性的分析，在正常草蝦及受到白點病毒的草蝦的比較分析上，提供了一個非常豐富的資源。除此之外，EST技術不但可以大大地幫助in silico上的分析以及試圖說明白點病毒的致病機轉外，還可以試圖找出草蝦受到白點病毒感染後可能的反應機制。這份研究也許於往後以全面性的角度來研究草蝦宿主與白點病毒之間的關係上，可提供相當重要的研究基礎。

White spot syndrome (WSS) is a viral disease that affects most of the commercially cultivated marine shrimp species, not only in Taiwan but globally. However, a little information is available based on molecular basis of the host-virus interaction. Previous studies have been reported, which focus on specific organ issues. Nevertheless, the effect of white spot syndrome virus (WSSV) of infection in shrimps is a systematic mat-ter. In this study, we used postlarvae of Penaeus monodon in attempt to observe global gene regulations between host and WSSV utilizing the expressed sequence tag (EST)-based approach. We performed an analysis of 13,969 high-quality ESTs sequenced from 3’ end of cDNA clones of two libraries constructed from normal and WSSV-infected postlarvae. The analysis includes comparison of ESTs with known functions of anno-tated genes in databases, identification of up or down regulated expressed genes in hosts after WSSV infection, and functional classification utiliz-ing Gene Ontology (GO) scheme and Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathways. In all, 46% of these ESTs were matched with known genes stored in National Center of Biotechnology Information (NCBI) non-redundant (nr) database, 44% were assigned with GO functions, and 31% were annotated with KEGG pathways. In conclusion, 13,969 high-quality ESTs generated from postlarvae of Pe-naeus monodon in this study provide a rich source for comparative analy-sis of normal and WSSV-infected shrimp’s biology. EST-based strategy not only greatly facilitates in silico expression profiling and elucidating WSSV pathogenesis, but also reveals shrimp’s response to the virus in-fection. This study may be of importance in a systematic view of regulation between shrimp hosts and WSSV.

中文摘要 I
ABSTRACT III
TABLE OF CONTENTS V
LIST OF FIGURES VII
LIST OF TABLES VIII
INTRODUCTION 1
GENERAL BACKGROUND INFORMATION 1
LITERATURE REVIEW 3
PURPOSE OF RESEARCH 4
SIGNIFICANCE OF RESEARCH 5
OVERVIEW 5
BACKGROUND 7
EXPRESSED SEQUENCE TAG 7
GENE ANNOTATION 8
SEQUENCE ASSEMBLY 9
REPEAT MASKING 9
GENE PREDICTION / FINDING 10
THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION 11
BASIC LOCAL ALIGNMENT SEARCH TOOL 12
IPROCLASS DATABASE 12
THE GENE ONTOLOGY 13
KYOTO ENCYCLOPEDIA OF GENES AND GENOMES 15
FISHER’S EXACT TEST 16
MATERIALS AND METHODS 19
SHRIMP, VIRUS AND CHALLENGE 19
RNA EXTRACTION AND CDNA LIBRARIY CONSTRUCTION 19
SEQUENCING 20
SEQUENCE ANALYSIS 21
FUNCTIONAL ANNOTATION AND EXPRESSION ANALYSIS 22
STATISTICAL EVALUATION OF EST OCCURRENCE 23
RESULTS 26
GENERATION AND ANALYSIS OF EST LIBRARIES 26
SEQUENCE SIMILARITY 29
FUNCTIONAL CLASSIFICATION BASED ON GENE ONTOLOGY 32
FUNCTIONAL CLASSIFICATION BASED ON KEGG PATHWAYS 39
IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES 41
DISCUSSION AND CONCLUSION 49
BIBLIOGRAPHY 53

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