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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張麗冠 | |
| dc.contributor.author | Kai-Li Wang | en |
| dc.contributor.author | 王開立 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:46:22Z | - |
| dc.date.available | 2021-10-17 | |
| dc.date.copyright | 2016-10-17 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-06 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78216 | - |
| dc.description.abstract | 白點症病毒 (white spot syndrome virus, WSSV) 為目前蝦養殖產業最主要也是最致命的病原菌。自白點症病毒爆發至今已有二十餘年，但因其開放譯讀區的序列與目前已知的物種有很大的差異，因此至今尚未能完全了解其致病機制並發展出有效的藥物來控制白點症病毒所造成的疫情。白點症病毒為一具有外套膜的大型雙股DNA病毒，能感染許多的甲殼類動物 (crustacean)，其中對蝦科 (Penaeidae) 在感染此病毒後會在二至五天內死亡，死亡率高達90%-100%。本研究室從白蝦 (Litopenaeus vannamei) 選殖出一轉錄因子，將此轉錄因子基因的胺基酸序列進行線上資料庫比對後，發現其為一個類Ying Yang 1 (YY1) 蛋白質，並將其命名為 LvYY1。先前本研究室的研究指出，LvYY1會透過結合於白點症病毒極早期基因wssv108以及ie1啟動子上的YY1結合位來調控啟動子的轉錄活性。Dorsal是哺乳類動物NF-κB家族轉錄因子中RelA的同源蛋白質，而許多報導指出，Dorsal轉錄因子會參與無脊椎動物的Toll/NF-κB調控路徑，此路徑為無脊椎動物先天性免疫的調控者。本研究透過冷光報導分析法 (luciferase reporter assay) 發現LvYY1能活化wssv108啟動子的轉錄活性至10.39倍，過量表現LvDorsal時則無法活化wssv108基因的表現。但是當共表現LvYY1與LvDorsal時會協同活化wssv108啟動子的轉錄活性至44.57倍，此現象說明LvYY1及LvDorsal會協同調控極早期基因wssv108的表現。除此之外，LvYY1與LvDorsal也會協同調控ie1及wssv304核心啟動子的轉錄活性。另外利用GST-pull down法發現LvYY1與LvDorsal能在細胞體外直接結合，而透過免疫共沉澱法也得知LvYY1分別能與LvDorsal及PmTBP於細胞內結合。然後以電泳泳動性轉移分析法 (electrophoretic mobility shift assay, EMSA) 及DNA親和性沉澱分析法 (DNA affinity precipitation assay, DAPA) 證實LvYY1可能會伴隨著細胞中內生的轉錄因子結合在wssv304的核心啟動子上。LvYY1於協同效應中扮演著關鍵的角色，而白點症病毒能夠挾持宿主轉錄因子LvYY1及LvDorsal大量表現極早期基因，以協助自身基因組的複製。 | zh_TW |
| dc.description.abstract | White spot syndrome virus (WSSV) is a major and deadly pathogen that causes heavy losses on the shrimp farming worldwide. It has been almost twenty years since its emergence in 1990s. Until now, there are no effective treatments for WSSV due to lacking of the sequenced and annotated genomes for the host species. WSSV is a large enveloped dsDNA virus. There are many crustacean host susceptible to WSSV, especially it causes the penaeidae death with a mortality rate of 90%-100% within five days. LvYY1 was cloned from Litopenaeus vannamei in our lab. According to the amino acid sequence blast on NCBI, we found that it is a YY1-like protein, thus it was named Litopenaeus vannamei YY1 (LvYY1). In recent studies, we showed that the LvYY1 promotes the transcription of the critical WSSV IE genes, ie1 and wssv108. Moreover, L. vannamei Dorsal (LvDorsal), a RelA (p65) homolog in the mammals, was known to participate in Toll/NF-κB pathway to regulate shrimp innate immune for pathogen resistance. In this study, we found that LvYY1 activates wssv108 promoter activity 10.39-fold in an insect cell model. However, overexpression of LvDorsal failed to activate wssv108 expression. Interestingly, GST-pull down and co-immunoprecipitation results indicate that LvYY1 interacts with LvDorsal and PmTBP respectively, and the two factors synergistically activate wssv108 promoter activity 44.57-fold in cotransfection experiments. We also found that LvYY1 and LvDorsal synergistically activate ie1 and wssv304 core promoter activity. Electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) results revealed that LvYY1 binds to wssv304 core promoter. Taken together, these results demonstrate that LvYY1 plays a key role in the synergistic effect. LvYY1 and LvDorsal synergistically modulate the transcription of wssv108, ie1 and wssv304, which may be a strategy employed by WSSV to enhance viral replication. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:46:22Z (GMT). No. of bitstreams: 1 ntu-105-R03b22040-1.pdf: 2455339 bytes, checksum: c46242e9e91437832db670a659b08ead (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 謝辭……………………………………………………………………………………… i
中文摘要 ii Abstract iii 第一章 前言 1 1. 臺灣蝦養殖產業的發展概況 1 2. 白點症病毒 (White spot syndrome virus, WSSV) 2 3. 白點症病毒的極早期基因 4 4. Ying Yang 1 (YY1) 轉錄因子 5 5. NF-κB 家族轉錄因子 7 第二章 材料與方法 9 1. 實驗菌種與細胞株 9 2. 質體DNA萃取 9 3. 質體建構 9 4. 勝任細胞的製備 10 5. 細胞轉型 11 6. 細胞轉染 11 7. 細胞核蛋白質萃取 12 8. 冷光報導活性分析 (Luciferase reporter assay) 12 9. 蛋白質的表現誘導 13 10. 西方點墨法 (Western blotting) 13 11. 電泳泳動性轉移分析 (Electrophoretic mobility shift assay, EMSA) 14 12. GST-pull down 15 13. 免疫共沉澱法 (Co-immunoprecipitation, Co-IP) 15 14. DNA親和性沉澱分析 (DNA affinity precipitation assay, DAPA) 16 第三章 結果 17 1. LvYY1與LvDorsal會協同活化wssv108啟動子 17 2. LvYY1會與LvDorsal在細胞內結合 17 3. LvYY1與LvDorsal在細胞體外直接結合 18 4. 縮減wssv108啟動子片段對於LvYY1與LvDorsal協同效應之影響 18 5. LvYY1與LvDorsal會協同活化wssv304的核心啟動子 18 6. LvYY1與LvDorsal對ie1的核心啟動子的協同活化 19 7. LvYY1與PmTBP在細胞內結合 19 8. LvYY1會結合於wssv304的核心啟動子 20 9. LvYY1結合於wssv304 啟動子上的TATA box及其後8-mer的序列 21 10. LvDorsal會結合於ie1啟動子上的NF-κB結合位 21 11. LvDorsal於協同效應中所扮演的角色 22 第四章 討論 24 第五章 圖表 30 表1、本研究所使用的引子列表 30 圖1、LvYY1與LvDorsal對於極早期基因wssv108啟動子轉錄活性的影響 34 圖2、LvYY1與LvDorsal在細胞內的結合 35 圖3、LvYY1 與 LvDorsal 在細胞外的結合 36 圖4、LvYY1與LvDorsal對於極早期基因wssv108啟動子片段轉錄活性的影響 38 圖5、LvYY1與LvDorsal對於極早期基因wssv304啟動子片段轉錄活性的影響 40 圖6、LvYY1 與 LvDorsal 對於極早期基因ie1啟動子片段轉錄活性的影響 42 圖7、LvYY1與LvDorsal協同活化極早期基因wssv304及 ie1的核心啟動子 43 圖8、LvYY1與PmTBP在細胞內的結合 44 圖9、西方點墨法偵測LvYY1及LvDorsal蛋白質在Sf9昆蟲細胞中的表現 45 圖10、LvYY1結合於wssv304核心啟動子上 46 圖11、以多點突變競爭者序列分析LvYY1的DNA結合區域 47 圖12、LvYY1與LvDorsal會結合於ie1啟動子上的YY1及NF-κB結合位 48 圖13、以電泳泳動性轉移法分析LvDorsal與wssv304核心啟動子的結合 49 圖14、以DNA親和性沉澱法分析LvDorsal與wssv304核心啟動子的結合 50 圖15、LvYY1及LvDorsal協同調控wssv304核心啟動子的模式圖 51 圖16、E. coli BL21(DE3) 所表現的LvYY1能結合於極早期基因ie1啟動子上的YY1結合位 52 第六章 參考文獻 53 第七章 附錄 65 | |
| dc.language.iso | zh-TW | |
| dc.title | 白蝦YY1及Dorsal蛋白質協同調控白點症病毒極早期基因的表現 | zh_TW |
| dc.title | Regulation of the immediate-early genes of white spot syndrome virus by Litopenaeus vannamei YY1 and Dorsal | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 劉宛菁,王涵青,陳慧文 | |
| dc.subject.keyword | White spot syndrome virus (WSSV),Litopenaeus vannamei,wssv108,ie1,wssv304,Ying Yang 1,Dorsal, | zh_TW |
| dc.relation.page | 67 | |
| dc.identifier.doi | 10.6342/NTU201600589 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-06 | |
| 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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