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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 羅秀婉(Show-Wan Lou) | |
| dc.contributor.author | Yu-Chi Li | en |
| dc.contributor.author | 李昱錡 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:41:42Z | - |
| dc.date.available | 2011-08-14 | |
| dc.date.copyright | 2008-08-14 | |
| dc.date.issued | 2008 | |
| dc.date.submitted | 2008-07-31 | |
| dc.identifier.citation | 曾文陽。1998。石斑魚養殖學。前程出版社。
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40139 | - |
| dc.description.abstract | 當病毒侵入宿主後，會利用各種機制去改變宿主基因的表現，造成宿主的某些基因表現量上升或下降，以利病毒在宿主體內繁殖；反之，宿主亦會表現許多基因對抗病毒的侵襲。近年來，由於神經壞死病毒(nervous necrosis virus, NNV)所引發病毒性神經壞死症(viral nervous necrosis disease, VNN disease)對養殖業造成重大損失。以石斑魚而言，神經壞死病毒造成白身幼苗前高致死率，而此病毒主要感染石斑魚的神經組織如腦、脊髓和視網膜，並呈現空泡化。為了研究病毒感染白身石斑魚苗基因表現的變化量，首先建立石斑魚之噬菌體 cDNA基因庫（λ phage cDNA library），轉換為質體載體後，挑選6000個選殖株用來構築基因微陣列晶片（microarray chip）。另外以NNV病毒顆粒、免疫刺激物lipopolysaccharide（LPS）和 poly inosinic:cytidylic 【poly (I:C)】，分別注射白身點帶石斑，每天萃取total RNA一共五天，製備雜合探針，藉由微陣列分析這些基因在不同刺激物及不同天數之表現變化來篩選受到調控之表現基因。
本實驗分析NNV感染石斑魚苗後至少一天具有2倍差異性表現基因，屬於促進（up- regulated）表現有361個，而抑制（down-regulated）表現有747個，比LPS注射後（140和359個）或poly(I:C) 注射後（305和452個）的基因數目還多，表示當病毒侵襲，的確會引發較多基因受到病毒調控或是宿主本身表現以對抗病毒感染。將促進表現差異大於2倍和抑制表現大於2.5倍之基因進行序列定序，再利用NCBI 資料庫比對分析。扣除重複總共分析出83個NNV病毒感染後具有差異性表現基因，其中52個基因屬於促進表現，31個基因屬於抑制表現。這些差異性表現的基因群包括：轉錄，轉譯，免疫，結構蛋白， 蛋白質降解，代謝，運送，連結蛋白（adhesion protein），生合成，緊迫（stress protein），鈣離子結合，催化，蛋白酶，細胞週期以及能量合成等。這些分析提供NNV病毒感染石斑魚苗分子層次的調控表現。 | zh_TW |
| dc.description.abstract | Following infection, viruses use various mechanisms to control host genes expression. Some of the host genes may be activated or suppressed. And it makes the host cell to be an appropriate place for virus propagation. Meanwhile, the host cells also express genes to against virus infection. Recently, viral nervous necrosis disease, nervous necrosis virus (NNV) causes a great damage to grouper aquaculture in Asia. In order to understand the characteristics of virus infection, we established a λ phage cDNA library of virus infected-host tissue, grouper brain tissue, to screen the differentially-express genes in response to NNV infection, and used 6000 candidates, to construct the microarray chip. The chips was then hybridized with probes prepared from NNV, LPS or poly (I:C) injected-grouper larvae, respectively.
After analyzing two fold changes in at least one of five days of microarray data, the gene numbers of NNV-infected grouper was more than that of LPS- and poly(I:C)-injected. It suggests that more genes were regulated when virus invasion. After comparing these genes sequences with NCBI genetic database, it was observed that these gene’s function can be categorized to transcription, structure, protein degradation, immune, metabolism, transport, protein-protein adhesion, biosynthesis, stress, calcium binding proteins, catalysis, protease, translation, energy or cell cycle. This information could be helpful to further understand the molecular mechanisms in NNV-host interaction and the development of control measures against NNV infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:41:42Z (GMT). No. of bitstreams: 1 ntu-97-R95b45024-1.pdf: 3023650 bytes, checksum: 7bb3ef48fdfa23ae752db570274cdf22 (MD5) Previous issue date: 2008 | en |
| dc.description.tableofcontents | 中文摘要………………………………………………………………I
英文摘要………………………………………………………………Ⅱ 目錄……………………………………………………………………Ⅳ 圖目次…………………………………………………………………Ⅵ 表目次…………………………………………………………………Ⅶ 壹、 前言………………………………………………………1 1.1 石斑魚之簡介與台灣養殖現況…………………………1 1.2 神經壞死病毒之特性與疾病症狀………………………3 1.3 差異性表現基因之篩選方法……………………………6 1.4 實驗目的…………………………………………………10 貳、 材料與方法………………………………………………12 2.1 GB3細胞之培養與NNV病毒之繁殖……………………………12 2.2 不同試劑注射石斑魚苗………………………………………13 2.3 石斑魚腦部組織cDNA基因庫之構築…………………………13 2.3.1 石斑魚腦部組織mRNA之萃取與RNA電泳……………16 2.3.2 cDNA之合成與修飾……………………………………17 2.2.3 分離不同大小之cDNA片段……………………………19 2.3.4 cDNA嵌入片段與載體之接合反應……………………20 2.3.5 λ噬菌體之包裝………………………………………20 2.3.6 宿主細胞之製備………………………………………21 2.3.7 石斑魚腦部組織cDNA基因庫效價之測定…………………21 2.4 轉換噬菌體之載體為質體載體………………………………21 2.5 基因微陣列之雜合反應………………………………………23 2.5.1 Total RNA之萃取、純化與RNA電泳…………25 2.5.2 基因微陣列晶片之前準備………………………26 2.5.3 cDNA合成與標定………………………………26 2.5.4 雜合反應…………………………………………27 2.6 差異性表現之基因分析………………………………………28 2.7 定序及序列分析………………………………………………28 參、 結果……………………………………………………30 3.1 石斑魚腦部組織 cDNA 基因庫之建立與效價………………30 3.2 基因微陣列雜合反應…………………………………………30 3.3 基因微陣列晶片之分析………………………………………31 3.4 差異性表現 cDNA 之序列分析………………………………32 肆、 討論……………………………………………………………………34 伍、 參考文獻………………………………………………41 陸、 圖………………………………………………………49 柒、 表………………………………………………………61 Figure Figure 1. Messenger RNA extracted NNV-infected grouper brain tissue at different period………………………………………………………………....….…49 Figure 2. Synthesis first- and second- strand cDNA..……………………..……..……50 Figure 3. Size fractionation of cDNA..………………….………………………….…51 Figure 4. Complementary DNA microarray of NNV-infected grouper brain tissue…..52 Figure 5. Complementary DNA microarray of LPS-injected grouper brain tissue...….53 Figure 6. Complementary DNA microarray of poly(I:C)-injected grouper brain tissue……………………………………………………...……………….. 54 Figure 7. Core reference set for two fold up-regulated gene expression..……………..55 Figure 8. Core reference set for two fold down-regulated gene expression…….…..…56 Figure 9. Functional categories of the differentially expression genes of NNV-infectd grouper brain tissue.…………………..……..………………………..……57 Figure 10. Groups construction of similar 2 fold up-regulated expression pattern from microarray analysis..………………………………….……………….……58 Figure 11. Groups construction of similar 2.5 fold down-regulated expression pattern from microarray analysis.…………………….……………………….……59 Figure 12. Cluster analysis of microarray expression using the expression ratio in NNV-infected grouper………………….....……………………..…………60 Table Table 1. List of NNV, LPS and poly(I:C) up-regulated genes more than 2 fold change.………………………………………………..…………………….61 Table 2. List of NNV, LPS and poly(I:C) down-regulated genes more than 2.5 fold change…………………………………………………………………....…64 | |
| dc.language.iso | zh-TW | |
| dc.subject | 神經壞死病毒 | zh_TW |
| dc.subject | 點帶石斑 | zh_TW |
| dc.subject | microarray | en |
| dc.subject | orange spotted grouper | en |
| dc.subject | nodavirus | en |
| dc.title | 神經壞死病毒調控石斑魚基因表現之研究 | zh_TW |
| dc.title | Gene expression profiling of nodavirus infected-orange spotted grouper Epinephelus coioides | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 96-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 張繼堯(Chi-Yao Chang) | |
| dc.contributor.oralexamcommittee | 林正輝(Cheng-Hui Lin) | |
| dc.subject.keyword | 神經壞死病毒,點帶石斑, | zh_TW |
| dc.subject.keyword | nodavirus,orange spotted grouper,microarray, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2008-08-01 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| Appears in Collections: | 漁業科學研究所 | |
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