- 學位論文
烏魚受奴卡氏菌感染之轉錄體分析
Transcriptome analysis of Mugil cephalus after challenge with Nocardia seriolae
摘要
烏魚(Mugil cephalus)是台灣經濟價值非常高的養殖物種，其中又以烏魚子最為人所熟知。在烏魚養殖的過程中，可能會因為高密度養殖或是季節變化，使得外來病原入侵。奴卡氏菌(Nocardia seriolae)是其中一種容易感染烏魚的細菌，魚隻會因為感染奴卡氏菌死亡，造成烏魚養殖的損失。為了全面瞭解烏魚被奴卡氏菌感染之後的免疫反應，且制定後續更有效的預防策略，我們透過Illumina轉錄體分析技術，用ex vivo的方式分離烏魚血液中的白血球，並且將烏魚白血球和奴卡氏菌共培養，而後進行轉錄體定序。 在Illumina轉錄體定序(n=3)完成之後，控制組及奴卡氏菌感染組總共6個樣本的序列會進行重新組裝(de novo assembly)，結果總共得到344,329個轉錄本，GC含量、N50、E90N50及平均長度分別為45%、1,738 bp、2,221 bp及928 bp。在經過序列相似度比較與篩選出最完整的相似序列後，總共獲得65,276個unigenes，並有53,620個unigene可注釋基因功能。透過BUSCO的3,640個單拷貝基因進行組裝完整度的評估，總共有74.4%的序列完全覆蓋。比較控制組與奴卡氏菌感染組的基因表現量後，總共得到225個具有表現差異的基因，其中包含140個正調控的基因及85個負調控的基因。比對COG、GO、KEGG及IPA等功能性資料庫，我們從225個表現差異基因中找到免疫相關的基因並且從中揭示出烏魚對抗奴卡氏菌的相關免疫途徑，其中包含IL-17信號通路(IL-17 signaling pathway)、白血球經內皮細胞層遷移(leukocyte transendothelial migration)、NOD-like受體信號通路(NOD-like receptor signaling pathway)。 此外，我們也透過體內實驗(in vivo)的方式進行奴卡氏菌攻毒實驗，使用qPCR實驗驗證轉錄體分析中具有表現差異的重要免疫基因IL17C、MMP-9、MMP-13、PANX1、F3、TNFRSF25、TNFRSF6B、NLRC3。 在本次的研究中，我們透過轉錄體分析的方式，更加了解烏魚在奴卡氏菌入侵後，參與進行調控的免疫基因及途徑，這個研究可以在未來針對奴卡氏菌在烏魚所引起的疾病，制定相關策略並進行預防。
並列摘要
Mugil cephalus (grey mullet) is an important fish species for cultivation in Taiwan. During a long cultivation period, intensive aquaculture or seasonal variations might cause the invasion of the pathogens. Nocardia seriolae is a gram-positive bacteria that might easily infect M. cephalus and cause mortality. Therefore, to comprehensively understand the immune response of M. cephalus after N. seriolae infection and formulate effective strategies in the future, Illumina RNA sequencing technology was applied in our study. RNA sequencing was executed with the leukocytes isolated from M. cephalus blood and co-cultured with N. seriolae. De novo assembly was conducted with six samples from control groups and N. seriolae infection groups. 344,329 transcripts were yielded, GC content, N50, E90N50, and the average length were 45%, 1,738 bp, 2,221 bp, and 928 bp, respectively. A total of 65,276 unigenes were obtained after optimization by TransDecoder and CD-HIT. Moreover, 74.4% of the sequences were completely recovered from the 3640 single-copy orthologs in BUSCO, and EggNOG annotated 53,620 unigenes. 225 differentially expressed genes were discovered, including 140 up-regulated and 85 down-regulated genes, with at least a two-fold difference between control and N. seriolae infection groups. After COG, GO, KEGG, and IPA analysis, we found immune-related genes among 225 differentially expressed genes (DEGs), which revealed the immune-related pathways of M. cephalus after N. seriolae infection, such as IL-17 signaling pathway、leukocyte transendothelial migration、NOD-like receptor signaling pathway. Furthermore, in vivo qPCR was used to confirm the results from RNA sequencing. The crucial DEGs such as IL17C, MMP-9, MMP-13, PANX1, F3, TNFRSF25, TNFRSF6B, and NLRC3 were randomly tested in qPCR. In this study, we assembled the transcriptome of M. cephalus and compared the gene expression profiles among control and N. seriolae infection groups to better understand the immune genes and pathways against bacteria invasion and frame possible strategies to prevent the outbreak of nocardiosis.
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