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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40498完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張麗冠 | |
| dc.contributor.author | Shu-Han Yang | en |
| dc.contributor.author | 楊舒涵 | zh_TW |
| dc.date.accessioned | 2021-06-14T16:49:23Z | - |
| dc.date.available | 2016-08-17 | |
| dc.date.copyright | 2011-08-17 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/40498 | - |
| dc.description.abstract | 白點症病毒 (white spot syndrome virus, WSSV) 感染蝦會導致高度的致死率，而本研究室先前在草蝦 (Penaeus monodon) 中成功選殖了第一個Kruppel-like factor (KLF)，若將KLF抑制則會增加被WSSV感染的蝦體存活率。經由基因體資料庫分析比對，發現草蝦和白蝦 (Litopenaeus vannamei) 彼此具有高度的基因相似性，因此根據草蝦KLF (PmKLF) 的序列，本研究在白蝦組織中也成功的選殖出KLF，並命名為LvKLF。LvKLF全長可以轉譯出360個胺基酸，經由序列分析發現草蝦和白蝦KLF胺基酸具有93%相似性，且鋅指區域完全相同，而利用抗KLF抗體能偵測到大小約為40 kDa的LvKLF。利用螢光顯微鏡觀察LvKLF在Sf9細胞表現時會在細胞核中聚集成點狀分布。之後進行病毒極早期基因整體分析，篩選了在白點症病毒台灣分離株基因體上編號分別為108、126、135、136、137、140、150、154、155、156、157、159、164、234以及304號的開放譯讀區 (open reading frame)，在白蝦體中利用siRNA抑制LvKLF基因表現後，再感染白點症病毒，於不同時間點收取蝦體組織直到感染病毒48小時，之後利用反轉錄聚合酶鏈反應分析，結果發現這十五個白點症病毒極早期基因表現均被抑制，另外以即時聚合酶鏈反應定量分析也得到相同的抑制結果。最後，進一步的在昆蟲細胞Sf9中，以預測的WSSV極早期基因啟動子進行報導基因分析，發現LvKLF會活化108、126、140、164、234、304號基因啟動子的活性。因此，本研究發現了白蝦的KLF對於蝦體在被WSSV感染的過程中扮演很重要的角色。 | zh_TW |
| dc.description.abstract | White spot syndrome virus (WSSV) infection can cause a significant mortality to shrimp cultivation. Our previous study identified the first kruppel-like factor (PmKLF) in Penaeus monodon. Inhibition of PmKLF by double-stranded RNAs decreased the mortality in WSSV-infected shrimps. According to genomic database, the sequences of P. monodon and Litopenaeus vanname display highly identity. In this study, a KLF from L. vannamei was cloned and characterized. The full-length LvKLF gene is 1080 bp, encoding a polypeptide of 360 amino acids. Sequence analysis showed that the identity of LvKLF and PmKLF amino acids is 93% and the zinc-finger DNA binding domain are the same. Immunoblotting using the anti-KLF antibody indicated that the apparent molecular mass of LvKLF protein is about 40 kDa. Moreover, immunofluorescence analysis revealed that LvKLF is located in the nucleus as dots after overexpressing LvKLF in Sf9 insect cells. So far, fifteen WSSV candidate immediate early (IE) genes including wssv108、wssv126、wssv135、wssv136、wssv137、wssv140、wssv150、wssv154、wssv155、wssv156、wssv157、wssv159、wssv164、wssv234 and wssv304 in the Taiwan isolate had been identified. This study indicated that these IE genes were inhibited in WSSV-challenged shrimps that had been injected with double-stranded RNAs of LvKLF. Furthermore, the reporter assay by using predictive WSSV IE promoters revealed that LvKLF enhanced the promoter activities of wssv108, wssv126, wssv140, wssv164, wssv234 and wssv304. Hence, this study demonstrates that the kruppel-like factor in Litopenaeus vannamei plays a crucial role in WSSV infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-14T16:49:23Z (GMT). No. of bitstreams: 1 ntu-100-R98b47404-1.pdf: 7441437 bytes, checksum: ca18526d04fcf8fa3eae6a942194cb1b (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 誌謝…………………………………………………………………………...………ii
中文摘要 ……………………………………………………………………………..iii Abstract …………….……………………………………………………………….iv 一. 前言 1 1. 台灣水產養殖現況 1 2. 白點症病毒 (White Spot Syndrome Virus, WSSV) 1 3. 白點症病毒的生活史 3 4. 白點症病毒的極早期基因 (Immediate early genes) 5 5. Kruppel-like factor (KLF) 7 二. 研究目的 13 三. 材料與方法 14 1. 菌種與細胞株 14 2. 質體的建構 14 3. 勝任細胞的製備 14 4. 大腸桿菌的轉型作用 14 5. 質體DNA的萃取 15 6. 蝦組織RNA的萃取 15 7. 白蝦KLF之基因選殖－5’端快速放大cDNA端點 15 8. 白蝦KLF基因之構築 16 9. LvKLF與PmKLF序列的分析比對 16 10. 反轉錄酶-聚合酶鏈反應 16 11. 大腸桿菌重組蛋白質His-LvKLF的誘導表現 17 12. 細胞轉染 18 13. 西方點墨法 18 14. 螢光染色分析 19 15. 冷光酵素活性分析 19 16. 雙股核醣核酸之製備 19 17. RNA靜默化試驗 20 18. 人工注射感染試驗與蝦組織檢體的收取 20 19. 即時聚合酶鏈反應 20 四. 結果 22 1. 白蝦LvKLF基因的選殖 22 2. 白蝦LvKLF和草蝦PmKLF的胺基酸序列比較 22 3. LvKLF於大腸桿菌、Sf9及白蝦體的表現 22 4. LvKLF會在細胞核內表現 23 5. LvKLF對白點症病毒極早期基因表現之調控分析 23 6. LvKLF會調控病毒極早期基因啟動子 25 7. LvKLF對白點症病毒極早期基因表現之調控分析 26 五. 討論 27 六. 圖表 33 表1、 本研究所建構的質體列表 33 表2、 本研究所使用的引子列表 41 表3、 LvKLF對於白點症病毒極早期基因預測啟動子區活性的調控 45 圖1、 LvKLF基因選殖的流程圖 46 圖2、 LvKLF的核酸序列和其對應的胺基酸序列 47 圖3、 LvKLF與PmKLF的胺基酸序列比對。 48 圖4、 重組LvKLF和白蝦內LvKLF的蛋白質表現 49 圖5、 LvKLF會在秋夜盜蛾 (Spodoptera frugiperda, Sf9)細胞的表現 50 圖6、 白蝦體內LvKLF基因靜默化會降低白點症病毒極早期基因表現 51 圖7、 利用基因靜默化抑制LvKLF基因會導致白點症病毒極早期基因wssv108、wssv126以及wssv140的表現量降低 52 七. 參考文獻 53 八. 附錄 68 附錄1、 哺乳類細胞Sp/KLF 家族的鋅指區域蛋白質序列比對 68 附錄2、 KLF分子結構示意圖 69 附錄3、 Sp1-like和KLF蛋白質的結構特性 70 附錄4、 KLF在白蝦 (L. vannamei) 血球細胞的表現位置 71 附錄5、白點症病毒極早期基因的預測啟動子序列 74 | |
| dc.language.iso | zh-TW | |
| dc.subject | KLF蛋白質 | zh_TW |
| dc.subject | 白蝦 | zh_TW |
| dc.subject | 白點症病毒 | zh_TW |
| dc.subject | 白點症病毒極早期基因 | zh_TW |
| dc.subject | Kruppel-like factor蛋白質 | zh_TW |
| dc.subject | WSSV immediate early gene | en |
| dc.subject | Kruppel-like factor | en |
| dc.subject | WSSV | en |
| dc.subject | Litopenaeus vannamei | en |
| dc.title | 白蝦 Kruppel-like factor於白點症病毒感染時之功能性探討 | zh_TW |
| dc.title | Role of the Kruppel-like factor in Litopenaeus vannamei after white spot syndrome virus infection | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王涵青,黃偉邦,呂健宏 | |
| dc.subject.keyword | 白蝦,白點症病毒,白點症病毒極早期基因,Kruppel-like factor蛋白質,KLF蛋白質, | zh_TW |
| dc.subject.keyword | Litopenaeus vannamei,WSSV,WSSV immediate early gene,Kruppel-like factor, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-12 | |
| 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 | |
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