斑馬魚HSC70啟動子作為脊椎動物模式創新冷誘導啟動子之研究

Abstract

為解決冬季環境低溫造成之養殖漁業損失，我們已成功轉殖鯉魚肌肉第三型肌酸激脢進入模式魚種並有效提高其低溫活動力與存活率。但使用CMV基因表現系統其持續大量之基因表現卻造成基因轉殖魚壽命中期後死亡率急速增加等負面影響，是故我們希望能發展一溫度調控之基因表現系統以符合應用環境之需要。 熱休克蛋白為一被廣為研究探討之逆境誘導基因，而在論文研究中發現其在許多物種中具有可被冷刺激誘導表現之特性。為了更深入了解其冷誘導表現特性與應用潛力，我們由模式魚種斑馬魚的基因中選殖出熱休克蛋白持續型-70之5’上游序列並使用螢光蛋白作為報導基因進行啟動子活性分析。在原生熱休克蛋白持續型-70之低溫誘導表現試驗，我們發現給予16度的低溫刺激5小時後，其mRNA表現量可大幅提高10倍。而將其上游2kb長度之啟動子區間選殖作為活性分析之表現質體，其基因轉殖斑馬魚在經過5小時的16度低溫誘導之後亦能呈2-3倍的mRNA增加量，螢光蛋白之表現在紫外光激發下亦可見明顯之增加，至此確定發現一脊椎動物冷誘導啟動子。而為了更加了解其啟動子調控功能，根據序列分析結果選殖不同刪除長度之啟動子。分別構築螢光蛋白報導系統進行起動子活性分析，結果發現其最短刪除區間約200bp長度之啟動子仍保有冷誘導能力。

This study reports the in vivo expression research of hsc70 gene in zebrafish and the cloning of zebrafish hsc70 promoter for temperature induction study, trying to establish a cold-inducible gene expression system to replace the constitutive CMV promoter system, which we used before in our cold tolerance project. Heat shock proteins (Hsps) are well-known stress-inducible chaperons. It had been reported can be induced by cold shock in many species from plant to vertebrate. To understand the regulation of cold-inducible gene expression, we identified the expression of zebrafish hsc70 by semi-quantitative real time-PCR, isolated the 5’-flanking sequence of zebrafish hsc70 gene and used a green fluorescent protein (GFP) for in vivo assay the promoter activity. In basal expression pattern assay, cold shock treatment resulted in a 10 folds enhanced induction of zebrafish hsc70 gene. In in vivo promoter assay, transient zebrafish showed an 2-3 folds enhanced induction. It’s clearly that we’ve identified a novel cold-inducible promoter in vertebrate. In order to further finding out the promoter function, a series deletion of the promoter region had been preformed. After in vivo expression assay we’ve found that the shortest promoter still keeps the cold-inducibility.