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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張麗冠 | |
| dc.contributor.author | Ting-Yi Huang | en |
| dc.contributor.author | 黃庭儀 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:12:14Z | - |
| dc.date.available | 2019-09-02 | |
| dc.date.copyright | 2014-09-02 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-19 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55982 | - |
| dc.description.abstract | Yin-yang 1 (YY1) 為一種廣泛表現於各組織之GLI-Krüppel家族的鋅指蛋白質 (zinc finger protein)，其特色為在蛋白質C端 (C-terminal) 具有一個Krüppel type與三個GLI type，共四個的鋅指元素，並會結合於保守性序列ATGG上，調節基因表現，在細胞中並扮演重要的調控角色。日前中國研究團隊自中國對蝦 (Fenneropenaeus chinensis) 中成功選殖出一轉錄抑制調控子基因，本研究根據序列比較與 RACE 分析，成功從白蝦 (Litopenaeus vannamei) 選殖出與其相似的基因序列。將該基因鋅指區胺基酸進行序列比對後發現與人類 (Homo sapiens)、斑馬魚 (Danio rerio)、果蠅 (Drosophila melanogaster) 的類YY1蛋白質鋅指區有96%的相似性，因此命名此基因為LvYY1。LvYY1的開放譯讀區全長1065核苷酸，並可轉譯出含有354個胺基酸的蛋白質產物。使用抗LvYY1抗體可以觀察到白蝦YY1蛋白質約位在65 kDa。透過免疫螢光分析可發現V5-LvYY1融合蛋白質在秋夜盜蛾細胞 (Spodoptera frugiperdam, Sf9) 中主要分布於細胞核內。進一步在果蠅細胞 (Schneider 2 cells, S2) 進行冷光報導分析 (reporter assay)，結果發現LvYY1會活化白點症病毒極早期基因ie1轉錄活性 (WSSV immediate early gene 1, ie1)，並以電泳流動性轉移分析 (EMSA) 證實LvYY1會結合在ie1啟動子上的ATGG序列形成蛋白質-DNA複合體。而在白蝦死亡率實驗中，LvYY1基因的表現若受到抑制，會使WSSV感染的白蝦呈現較低的死亡率。由本研究發現，LvYY1可結合於極早期基因ie1的啟動子上調控基因表現並影響白點症病毒的感染。 | zh_TW |
| dc.description.abstract | Yin-yang 1 (YY1) is an ubiquitously expressed GLI-Krüppel family of zinc finger protein. The protein has one KLF-type and three GLI-type zinc finger motifs at the C-terminal, binding to the consensus sequence ATGG, and regulating gene expression. China researcher recently cloned a transcriptional repressor gene from Fenneropenaeus chinensis. According to the gene sequence blast and RACE analysis, a similar gene sequence was cloned from Litopenaeus vannamei. The zinc finger domain of the gene showed 96% identical to human, zebrafish, and flies YY1-like genes, thus it was named LvYY1. The ORF sequence of LvYY1 is 1065 bps, encoding a polypeptide of 354 amino acids. Western blot analysis showed that L. vannamei endogenous YY1 protein migrated as 65 kDa and was recognized by anti-YY1 antibody. Immunofluorescence analysis showed that LvYY1-V5 was located mainly in the nucleus in Sf9 cells. Additionally, LvYY1 activated the expression of white spot syndrome virus immediate early gene wssv126 (ie1) in S2 cells by reporter assay. Moreover, electrophoretic mobility shift assay (EMSA) demonstrated that LvYY1 binds to the sequence ATGG in the ie1 promoter. Knockdown of the expression of LvYY1 transcript in WSSV-infected shrimp caused delayed cumulative mortality. Thus, LvYY1 can bind to WSSV ie1 promoter, which is critical to WSSV infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:12:14Z (GMT). No. of bitstreams: 1 ntu-103-R01b22042-1.pdf: 3434651 bytes, checksum: d412d5c465e55f8292bbaa071f353d6d (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii 第一章 前言 1 1. 臺灣蝦產業歷史與現況 1 2. 白點症病毒 (White Spot Syndrome Virus, WSSV) 2 3. 白點症病毒極早期基因 4 4. 鋅指蛋白質與Ying Yang 1 (YY1) 6 第二章 材料與方法 9 1. 菌種、細胞株與動物 9 2. 快速放大 cDNA 端點-白蝦YY1基因分析 9 3. 質體建構 9 4. 勝任細胞製備 10 5. 細胞轉型 10 6. 質體萃取 11 7. 蛋白質純化 11 8. 抗體製備 12 9. 細胞轉染 13 10. 核蛋白萃取 12 11. 西方墨點法 13 12. 免疫螢光染色 14 13. LvYY1結合位預測 14 14. 冷光酵素報導分析 15 15. 電泳游動性轉移分析 (EMSA) 15 16. 雙股核醣核酸製備 16 17. RNA 靜默 (RNA inference, RNAi) 17 18. 蝦組織RNA萃取 17 19. 反轉錄酶-聚合酶鏈反應 (RT-PCR) 18 20. 白蝦死亡率分析 19 第三章 結果 20 1. 白蝦LvYY1的序列分析 20 2. LvYY1基因在白蝦各組織中的表現情形 20 3. 抗 LvYY1抗體 (a-YY1) 的抗原製備 21 4. LvYY1蛋白質表現 22 5. LvYY1蛋白質在細胞中的分布 22 6. LvYY1基因抑制對於白點症病毒極早期基因ie1表現的影響 23 7. LvYY1調控白點症病毒極早期基因ie1表現 23 8. LvYY1結合在極早期基因ie1啟動子上的YY1預測結合位 24 9. 抑制白蝦LvYY1基因會減少白點症病毒的感染能力與毒性 25 第四章 討論 26 第五章 圖表 32 表一、文章中所用引子序列 32 Figure. 1. 白蝦LvYY1基因序列與鋅指區胺基酸序列比較 33 Figure. 2. LvYY1基因在各組織的表現 36 Figure. 3. 抗LvYY1抗體之抗原LvYY1-dZ蛋白質製備與檢測 37 Figure. 4. 西方墨點法偵測LvYY1蛋白質在E. coli、Sf9與白蝦中的表現 39 Figure. 5. LvYY1蛋白質在Sf9細胞中的分布 40 Figure. 6. 白蝦LvYY1基因抑制能減少白點症病毒極早期基因ie1的表現 41 Figure. 7. LvYY1活化白點症病毒極早期基因ie1表現 42 Figure. 8. LvYY1結合在ie1基因啟動子上 44 Figure. 9. 抑制白蝦LvYY1基因表現對白點症病毒感染所造成的存活率觀察 45 Figure. 10. GFP-LvYY1-dZ重組蛋白質在Sf9細胞中的分布 46 Figure. 11. 白蝦LvYY1基因在白點症病毒感染後的表現 47 第六章 參考文獻 48 | |
| dc.language.iso | zh-TW | |
| dc.subject | Yin-yang 1蛋白質 | zh_TW |
| dc.subject | 白點症病毒 | zh_TW |
| dc.subject | 南美白對蝦 | zh_TW |
| dc.subject | white spot syndrome virus (WSSV) | en |
| dc.subject | Yin-yang 1 | en |
| dc.subject | Litopenaeus vannamei | en |
| dc.title | 白蝦YY1蛋白質調控白點症病毒極早期基因wssv126 (ie1) 的表現 | zh_TW |
| dc.title | Regulation of the wssv126 (ie1) of white spot syndrome virus by Litopenaeus vannamei YY1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃偉邦,王涵青,劉宛菁,呂健宏 | |
| dc.subject.keyword | Yin-yang 1蛋白質,白點症病毒,南美白對蝦, | zh_TW |
| dc.subject.keyword | Yin-yang 1,white spot syndrome virus (WSSV),Litopenaeus vannamei, | en |
| dc.relation.page | 56 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-19 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-103-1.pdf 未授權公開取用 | 3.35 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。