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研究生:吳明鴻
研究生(外文):Ming-Hung Wu
論文名稱:巨大細胞病毒屬及蛙病毒屬之石斑虹彩病毒感染點帶石斑魚基因表現轉錄體之初步研究
論文名稱(外文):Preliminary transcriptomic analysis of gene expression in orange-spotted grouper (Epinephelus coioides) infected with grouper iridoviruses of Megalocytivirus and Ranavirus
指導教授:周信佑周信佑引用關係
指導教授(外文):Hsin-Yin Chou
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:99
中文關鍵詞:石斑魚巨大細胞病毒屬蛙病毒屬基因轉錄體定序數位基因表現
外文關鍵詞:GrouperMegalocytivirusRanavirusTranscriptomeRanavirus
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感染台灣養殖石斑魚的重要虹彩病毒有兩類,分別歸屬於巨大細胞病毒屬之TGIV (Grouper Iridovirus of Taiwan, TGIV) 以及歸屬蛙病毒屬之GIV (Grouper Iridovirus, GIV),兩者均會造成多種養殖魚類的全身性感染。本研究利用轉錄體定序技術 (Transcriptome) 及數位基因表現 (Digital Gene Expression, DGE) 分析,探討兩類虹彩病毒感染點帶石斑魚脾臟的基因表現。結果與未感染魚相比較,表現差異達四倍以上的基因,在感染TGIV魚的脾臟轉錄體中有204個基因為上升表現及713個基因為下降表現;而感染GIV魚的脾臟中則有641個基因為上升表現及829個基因為下降表現。進一步選擇血紅蛋白基因 (Hemoglobin subunit beta-2, Hb)、CC motif chemokine 19 precusor (CCL pre-19)、Toll-like receptor 9 isoform A、B (TLR9-A、B) 和Mx五個目標基因,以即時定量PCR (QPCR) 分析病毒感染以及疫苗免疫魚脾臟和頭腎中基因表現的變化情形。結果顯示,Hb基因在TGIV感染後第三天於頭腎中誘發表現,GIV感染後則無顯著差異,而免疫後攻毒組中,Hb於TGIV攻毒後直到第10天才被誘發,GIV攻毒後無顯著差異。CCL pre-19在脾臟中的表現均為先升後降,頭腎中則在感染TGIV後第3天誘發表現,而免疫後攻毒所誘發的表現量降低。TLR9-A、B在脾臟的表現量高於頭腎,兩病毒免疫後攻毒組的表現量皆低於對照組。另外,兩病毒感染後都會誘發Mx基因表現,免疫後攻毒組中,TGIV感染後第三天的表現量明顯高於其他各組。根據以上結果推論,Hb在兩病毒感染魚的誘發程度不一,可能與病毒感染後所造成的缺氧情形、脾臟腫大程度不同有關; CCL pre-19 即時調控著病毒感染後的反應;免疫後再次攻毒使得TLR9-A、B基因誘發程度降低;Mx基因在兩屬虹彩病毒感染後所造成不同的趨勢可能與所參與的路徑有所不同。此結果將幫助我們了解點帶石斑魚宿主與虹彩病毒病原的相互關係,也有助於發展對於控制虹彩病毒感染的策略。
Two iridoviruses, grouper iridovirus of Taiwan (TGIV) of genus Megalocytivirus and grouper iridovirus (GIV) of genus Ranavirus, have caused serious epidemics in several maricultured fish species in Taiwan. In this study, we used de novo transcriptome analysis and Digital Gene Expression (DGE) technology to investigate the gene expression profile changes in orange-spotted grouper (Epinephelus coioides) spleens after TGIV and GIV infections, separately. After DGE, the differentially expressed genes were identified using a 4-fold-change cutoff. Two hundred and four genes were found to be up-regulated in the spleen from TGIV-infected grouper, and 713 genes were down-regulated; in GIV-infected spleen, 641 genes were up-regulated and 829 down-regulated. Five genes, including hemoglobin subunit -2 (Hb), CC motif chemokine 19 precursor (CCL pre-19), Toll-like receptor 9 isoform A, B (TLR9-A, B) and Mx, were selected for further study. QPCR was performed to analyze their expressions in both spleen and headkidney from iridoviruse-infected groupers and from groupers that had been vaccinated and then was challenged with iridoviruses. At 3 days post-infection (dpi), the expression of Hb gene was induced in TGIV-infected but not in GIV-infected headkidney; vaccination delayed the induced expression of Hb in TGIV. CCL pre-19 had similar expression patterns between TGIV- and GIV-infected spleens: up-regulated at 1 dpi and down-regulated thereafter; in headkidney, TGIV induced CCL pre-19 expression at 3 dpi. Vaccination decreased the induced expression of CCL pre-19. For TLR9-A and B, after viral infections, the induced expression levels in spleen were higher than that in headkidney; after vaccination, the expression levels in viral-infected groupers were lower than that in uninfected grouper. Both viruses could induce Mx gene expression; after vaccination, the expression levels in TGIV-infected grouper at 3 dpi were significantly higher than that in GIV-infected grouper. Based on our results, we concluded that: differential expression levels of Hb gene might be related to anoxia and splenomegaly; CCL pre-19 regulated the immediate immune responses to viral infections; vaccination could reduce the induced expression levels of TLR9-A and B genes; different expression patterns of Mx gene between TGIV- and GIV-infected groupers suggested that distinct signal pathways are involved. These results could help us to have better understanding of the host–pathogen interactions in E. coioides, and to the development of control strategies against iridoviruses infection.
謝辭………………………………………………………………………i
中文摘要………………………………………………………………iii
英文摘要………………………………………………………………v
目錄……………………………………………………………………vii
圖目錄…………………………………………………………………x
表目錄…………………………………………………………………xi
前言………………………………………………………………………1
文獻整理…………………………………………………………………3
一、虹彩病毒科之簡介及分類依據…………………………………3
二、魚類虹彩病毒之簡介……………………………………………5
三、硬骨魚類之免疫系統概述………………………………………7
四、全基因轉錄體定序技術的發展與應用…………………………10
五、相關基因功能簡介………………………………………………13
材料與方法………………………………………………………………17
一、虹彩病毒感染石斑魚隻病毒液收集……………………………17
(一) 實驗材料…………………………………………………………17
1、實驗細胞株與病毒……………………………………………17
2、虹彩病毒………………………………………………………17
(二) 研究方法…………………………………………………………17
1、細胞培養………………………………………………………17
2、病毒製備………………………………………………………19
二、單位體積內病毒之計數…………………………………………20
(一) 實驗材料…………………………………………………………20
1、QPCR引子……………………………………………………20
2、質體DNA……………………………………………………20
3、QPCR儀器與相關藥品………………………………………20
(二) 研究方法…………………………………………………………20
1、絕對定量………………………………………………………21
三、感染TGIV或GIV病毒後各臟器之病毒量評估………………22
(一) 實驗材料…………………………………………………………22
1、實驗魚隻與病毒……………………………………………22
(二) 研究方法…………………………………………………………23
1、實驗設計……………………………………………………23
四、石斑魚感染虹彩病毒於不同稀釋倍率之死亡率評估…………23
(一) 實驗材料…………………………………………………………23
1、實驗魚隻與病毒……………………………………………23
(二) 研究方法…………………………………………………………23
1、實驗設計……………………………………………………23
2、軟體製作……………………………………………………24
五、Transcriptome 與Digital Gene Expression (DGE)………………24
(一) 實驗材料…………………………………………………………24
1、實驗魚隻與病毒…………………………………………24
(二) 研究方法…………………………………………………………24
1、實驗設計……………………………………………………24
六、即時定量PCR (QPCR)……………………………………………25
1、實驗魚隻與病毒…………………………………………25
2、多重相口服DNA疫苗………………………………………25
3、QPCR儀器與相關藥品………………………………………25
(二) 研究方法…………………………………………………………25
1、實驗設計……………………………………………………25
結果……………………………………………………………………….28
討論……………………………………………………………………….33
參考文獻…………………………………………………………………40
圖…………………………………………………………………………63
表…………………………………………………………………………77
附錄………………………………………………………………………82
圖目錄
圖一、 感染TGIV 或GIV後各臟器之病毒含量估…………………..63
圖二、 石斑魚感染TGIV於不同稀釋倍率之累積死亡率……………64
圖三、 石斑魚感染GIV於不同稀釋倍率之累積死亡率…………65
圖四、 基因轉錄體分析後的經由NCBI BLAST比對NR蛋白質資料庫的分圖………………………...………………………………66
圖五、 基因轉錄體分析之GO (Gene ontology) 功能性注釋……67
圖六、 基因轉錄體分析之COG (Clusters of Orthologous Group) 同源性族群析……………………………………………………68
圖七、 感染TGIV或GIV後之表現差異基因數 (DGE分析)…69
圖八、 感染TGIV與GIV後之表現差異基因數(四倍以上差異)……70
圖九、 五個目標基因於各採樣天數的RPKM表現情形 (DGE分析).71
圖十、 Hb基因於各採樣天數(多重相口服DNA疫苗攻毒組增加第10天採樣) 與點帶石斑魚EF1-α的相對表現量………………72
圖十一、 CCL pre-19基因於各採樣天數(多重相口服DNA疫苗攻毒組增加第10天採樣) 與點帶石斑魚EF1-α的相對表現量…..73
圖十二、 TLR9-A基因於各採樣天數(多重相口服DNA疫苗攻毒組增加第10天採樣) 與點帶石斑魚EF1-α的相對表現量..……74
圖十三、 TLR9-B基因於各採樣天數(多重相口服DNA疫苗攻毒組增加第10天採樣) 與點帶石斑魚EF1-α的相對表現量……75
圖十四、 Mx 10-3基因於各採樣天數(多重相口服DNA疫苗攻毒組增加第10天採樣) 與點帶石斑魚EF1-α的相對表現量……76
表目錄
表一(a)、基因轉錄體分析(感染魚與未感染魚之脾臟總體積cDNA)
組裝產量統計…………………………………………………77
(b)、基因轉錄體分析 (注釋結果統計)……………………………77
表二、TGIV感染後相較於GIV感染後脾臟中顯示上升 (Up-regulated)
的基因…………………………………………………………78
表三、TGIV感染後相較於GIV感染後脾臟中顯示下降
(Down-regulated) 的基因…..…………………………………79
表四、本研究所使用之特定引子……………………………………81
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