博士 / 國立臺灣大學 / 漁業科學研究所 / 103 / Orange-spotted grouper (Epinephelus coioides) with protogynous hermaphroditic features are one of the most economically important aquaculture species in Taiwan. However, larvae stage grouper are susceptible to infection by the bacterial pathogen Vibrio alginolyticus. To better understand the molecular mechanisms of the immune response to V. alginolyticus in Epinephelus coioides larvae, we used high-throughput deep sequencing technology to study the effect of infection on gene expression. A total of 114,851,002 reads were assembled, consisting of 9,687,355,560 nucleotides; these were further assembled into 209,082 contigs with a mean length of 372 bp. Gene ontology (GO) analysis of the transcriptome revealed 12 cellular component subcategories, 16 molecular function subcategories, and 42 biological process subcategories (P value < 0.05). A total of 32,664 Epinephelus coioides genes were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG); 1,504 differentially expressed genes (DEGs) were subsequently identified, in 12 categories (P value < 0.05). Vibrio infection affected the expression of genes involved in complementation, coagulation cascades, pathogen (Staphylococcus aureus) infection, phagosome activity, antigen processing, and the antigen presentation pathway. We conclude that the complement pathway of innate immunity and the hepicidin antimicrobial peptide may play important roles in the defense of Epinephelus coioides larvae against V. alginolyticus, and the immune response may activate at 4h after bacterial infection. These results implicate the complement pathway signal pathway in immunity during V. alginolyticus infection at early developmental stages, enhancing our understanding of the mechanisms underlying the immune response to Vibrio infection in Epinephelus coioides.
To realize other kind of grouper, we choose Epinephelus lanceolatus which were higher economic value then Epinephelus coioides, we have established and conducted first-round annotation of transcriptome profiles in V. alginolyticus -infected and non-infected Epinephelus lanceolatus larva. The result shown that total read value became to 28,705,411, 2,152,905,850 total base, 100,848 unigenes number, 5,913 unigenes number filter for FPKM>0.3, 2FC, p<0.05. On the GO analysis, total 30 GO numbers in cellular component, both biological process and molecular function are 58 GO numbers. We choose the cluster of immune pathway, the cluster of biological process contains 27 unigenes in immune response, 31 unigenes in immune system process, 9 unigenes in inflammatory response, and 43 unigenes in response to stress. Cellular component contain 8 unigenes in membrane attack complex. Both chemokine activity and chemokine receptors binding contain 9 unigenes in molecular function, cytokine activity obtained 13 and cytokine receptor binding contain 12 unigenes. On the KEGG analysis, contain 47 total pathways. Because of KEGG pathway involved lower then GO analysis, only include 1-4 genes. Finally we choose GO analysis became a main sign decided the pathway by GeneSpring software. The result of Real-time qPCR shown that TLR5, IL-1β, IL-8, SAA and hepcidin consisted strong expression between 4 to 16h, and indicate that V. alginolyticus probably stimulate TLR5 activity by bacterial flagellum, through by MyD88 dependent pathway, finally produce JunB, IL-1βand IL-8 from NFκB pathway induce pro-inflammatory effects or chemotactic effects. Otherwise possibility via by serum amyloid A stimulate neutrophils to infect tissue secret MMP9 then cleave IL-8 and to activate. IL-8 would enhance neutrophil chemotaxis. The express gene from C3, C6, C7, C8 and C9 indicate they would induction complement system and become a membrane attack complex to lysis bacteria membrane. Antimicrobial peptide contain highly expression possibility destroy the V. alginolyticus. The result indicate that immune pathway of V. alginolyticus infected Epinephelus lanceolatus probably via TLR5 regulate down- stream cytokine gene expression. Summarize two species of grouper, Epinephelus coioides infected by V. alginolyticus stimulated complement system and hepcidin expression, in Epinephelus lanceolatus both above-mentioned immune pathway also activation. Moreover, V. alginolyticus probably stimulated TLR5 by flagellum to mediate cytokine and inflammatory response.
| Identifer | oai:union.ndltd.org:TW/103NTU05451021 |
| Date | January 2015 |
| Creators | Yi-Da Wang, 王亦大 |
| Contributors | Jyh-Yih Chen, 陳志毅 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 112 |
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