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標題: | 草蝦熱休克同源蛋白七十基因啟動子之選殖與表現分析 Cloning and Expression Analysis of Heat Shock Cognate 70 Gene Promoter in Tiger Shrimp (Penaeus monodon) |
作者: | Kuo-Hung Chuang 莊國鴻 |
指導教授: | 宋延齡(Yen-Ling Song) |
關鍵字: | 草蝦,熱休克,熱休克蛋白七十,熱休克同源蛋白七十,啟動子,熱休克,基因, heat shock cognate 70,heat shock protein 70,hsp70,hsc70,tiger shrimp,Penaeus monodon,crustacean,promoter,chaperon,heat shock,stress, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 物種間皆極為保守的熱休克蛋白七十,對於細胞在正常的生理過程中,協助新生成的蛋白質做正確的構形摺疊,避免錯誤而使蛋白質功能失常;或是細胞處於環境壓力下(如:高溫),將遭受破壞的蛋白質予以修復,扮演要角。其表現調控以轉錄階段調控最為重要。本論文進行草蝦熱休克同源蛋白七十 (heat shock cognate 70, hsc70) 啟動子的研究。首先利用即時定量聚合酶鏈反應 (Real-time PCR),偵測草蝦血球中hsc70 訊息核苷酸 (mRNA) 在熱處理一小時,可提升8倍表現量,發現 hsc70 為一熱緊迫下增強表現啟動子。根據前人已發表之草蝦hsc70 基因,由草蝦基因體基因庫 (genomic DNA library) 中選殖出完整基因及啟動子。分析此選殖出的基因結構,發現草蝦hsc70基因含有兩個外插子(exon),唯一的一個內插子 (intron) 長度為1.5 kb,第一個外插子不載碼,而5端上游序列約3 kb。分析約1.1 kb長度啟動子區域預測出數種重要element,包含HSE、CAAT、SP1、core promoter region,並在intron內發現連續14個G序列。將啟動子接於pEGFP-1報導質體,證實 hsc70 啟動子可於轉染入秋行軍蟲卵巢細胞株Sf21後驅動綠螢光之產生;進一步接於 pGL3-Basic定量冷光報導質體,進行啟動子不同序列長度刪除實驗,初步找出相對於轉譯起始點上游 -1994 ~ -2160 bp內可能存在suppressor element;而-1799 ~ -1994或-1544 ~ -1780 bp的刪除,會嚴重降低啟動子之表現。在外部因子刺激啟動子表現的研究中:轉染hsc70 啟動子驅動之冷光報導質體於Sf21細胞後,再以AcMNPV感染細胞,可增強其報導質體之表現;胺基酸類似物及砷化物個別刺激下,僅砷化物可增強hsc70啟動子驅動報導質體之表現。這些結果顯示,選殖出的草蝦hsc70啟動子,有成為誘導型啟動子,用於轉殖蝦使用之潛能。 The tiger shrimp (Penaeus monodon) heat shock cognate 70 (hsc70) gene promoter was cloned for its further application in transgenic shrimps. The expression of the hsc70 mRNA increases 8 fold significantly after heat shock of tiger shrimps for 1 hour. In further study, this heat inducible hsc70 gene promoter was cloned from a tiger shrimp genomic DNA library and a 3 kb 5’ upstream sequence of the hsc70 gene was obtained. In transient gene expression assay, insertion of hsc70 promoter into pEGFP-1 reporter plasmid caused EGFP expression in transfected insect ovary Sf21 cell line. In the 1.1 kb promoter region, several potentially important elements were indetified such as putative CAAT, SP1, HSE, core promoter domain and a polypurine stretch site. Deletion analysis of the hsc70 promoter between -2160 ~ -2644 did not result in any diminution of reporter activity, while deletion of the region between -1799 ~ -1994 or -1544 ~ -1780 bp caused very significant decrease in the promoter activity. Infection of Sf21 cells with AcMNPV could enhance hsc70 promoter activity. Two amino acid analogues and arsenic acid were added to culture medium to test its influence to the promoter and the promoter activity could be enhanced in longer incubation time using arsenic acid at concentration between 0.4 to 1.2 mM, but not amino analoques. This result suggests that the cloned shrimp hsc70 gene promoter is inducible by heat、virus and heavy metal, so it may have a potential to be applied in transgenic shrimp as an inducible promoter. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31738 |
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顯示於系所單位: | 動物學研究所 |
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