臺灣博碩士論文加值系統

恆河稻田魚為廣鹽性魚類，能在廣泛的溫度與鹽度下生存，是一個了解魚類的環境適應反應的理想研究材料。本研究將恆河稻田魚腦組織在低溫以及不同鹽度下馴化一周後，使用 RNA 次世代定序收集序列進行轉錄體分析。RNA-Seq 資料進行轉錄體完全組裝、差異表現基因的分析、功能性的註解以及代謝途徑分析。從差異表現分析結果顯示，常溫下淡水處理下 prl-1 與其他 13 個在腦部組織具顯著上升表現，也認這些基因可能是恆河稻田腦部參與滲透壓調節機制的關鍵。此外，不同低溫下差異表現基因結果結合 KEGG (Kyoto Encyclopedia of Genes and Genomes) 途徑分析結果顯示，腦對於不同程度低溫共同涉及 45 個代謝途徑。其中認為包含 MAPK 訊息傳遞、鈣離子訊息傳遞、神經受體作用與細胞附著等途徑是腦部面臨低溫逆境主要的反應途徑。為了進行全生物體的同源基因比較，我們以有較良好基因註解的日本稻田魚的基因為基礎，在恆河稻田魚轉錄體中找出同源基因；根據演化壓力分析結果挑選出 123 個選擇壓力較高的基因，其中 5 個為已知基因。這些可提供作為未來探討稻田魚的種化關鍵的候選基因。本研究有助於對魚類面臨低溫逆境的調控機制提出具有前瞻性的新觀點，並可做為水產養殖產業在發展低溫逆境養殖技術的學理基礎。最後，本研究也將分析成果建立一個轉錄體分析資料庫平台，可供未來相關研究共享，網址為 : http://140.120.209.83/guan_test_templet/。

Euryhaline brackish medaka, Oryzias dancena, is able to survive under wide ranges of environmental temperatures and salinities, and thus becomes an ideal experimental animal for environmental adaption research. In this study, seawater (SW)- and fresh water (FW)-acclimated brackish medaka were separately transferred into different low temperatures for one week and then RNA-sequencing (RNA-Seq) of the brain transcriptome was conducted. The RNA-Seq data were then used for de novo assembly, differential expression analysis, functional annotation and pathway analysis. The results of differential expression analysis showed that prl-1 and some other 13 unknown genes are significantly differentially expressed in the brain tissue of the FW/normal-temperature group and these genes could participate in the modulatory mechanisms of osmoregulation in brains of the brackish medaka. In addition, 45 common KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of these differentially expressed genes under different low temperatures are involved. Among them, the MAPK signaling, calcium signaling, neuroactive ligand-receptor interaction, and cell adhesion molecules are considered as the main pathways in brain under low-temperature stress. We also conducted a genome-wide selection pressure analysis between two closely related medakas, O. dancena and Oryzias latipes. According to the results of the analysis, 123 genes were under positive selection and only 5 of them were known. These candidate genes may provide valuable clues for medaka speciation researches. This study provides new insights into the regulatory mechanisms of fish in response to low temperatures of environments. The results can also be used as the basis of aquaculture industry in developing low-temperature adversity culture technology. Finally, a website (http://140.120.209.83/guan_test_templet/) based on the analyzed data of this study was also set up

中文摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vi
英文名詞與縮寫對照表 1
前言 2
一、 恆河稻田魚 2
二、 魚類對於逆境的生理反應 3
三、 魚類相關低溫研究 4
四、 次世代定序技術與轉錄體的應用簡介 5
(一) 轉錄體 5
(二) 定序技術簡介 7
五、 轉錄體分析 9
(一) 轉錄體組裝 10
(二) 基因表現量分析 12
(三) 差異表現統計 15
六、 基因註解 16
(一) 生物資料庫 19
七、 富集分析 (Enrichment analysis) 21
八、 演化壓力 22
研究動機 23
材料方法 25
一、 實驗環境與實驗動物與定序 25
二、 轉錄體組裝 26
三、 基因表現量分析 27
四、 差異表現量分析 27
五、 TransDecoder進行 CDS 預測與同源基因搜尋比對 28
六、 整合整轉錄體基因註解資料 29
七、 富集分析 30
八、 富集分析資料彙整以及代謝途徑繪圖 31
九、 演化壓力計算 (Ka/Ks) 31
十、 網頁架構 32
結果 33
一、 轉錄體資料 33
二、 對應 Oryzias dancena 驗證序列來評估轉錄體 33
三、 與 Oryzias latipase 之間演化壓力分析 34
四、 基因表現量分析 35
五、 基因註解 36
六、 富集分析 37
七、 網頁呈現 40
討論 41
一、 轉錄體組裝結果評估 41
二、 與日本稻田魚 (O. latipase) 之間演化壓力分析 42
三、 基因表現量分析 43
四、 基因註解 44
五、 富集分析 45
六、 基因途徑分析 46
結論 52
參考文獻 88

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