白植友:斑马鱼磷脂醯丝胺酸受器启动子之选殖与功能分析

白植友:斑马鱼磷脂醯丝胺酸受器启动子之选殖与功能分析 白植友,斑馬魚磷脂醯絲胺酸受器啟動子之選殖與功能分析 中文摘要 磷脂醯絲胺酸受體 (Phosphatidylserine Receptor, PSR) 具有辨識凋亡 小體(apoptotic body)的功能與吞噬的能力,但是其如何動態現再斑馬魚 胚胎內尚未清楚。因此本論文將選殖PSR啟動子並與報導基因(EGFP)接 合,以便在斑馬魚胚胎發育時直接動態觀察PSR在不同時間與空間內之表 現。首先,我們證實斑馬魚PSR上游4 kbps序列具有啟動子區域,可以在斑 馬魚胚胎中啟動綠螢光基因。在PSRP-4K-EGFP基因轉殖魚發現,綠色螢 光基因在卵子時期到孵出時期皆有表現,主要在體節、眼、腦部、心臟、 孵化腺與泌尿道開口,皆有明顯的綠色螢光表現。利用此綠色螢光報導系 統,我們首次發現於受精後14天後的幼魚骨骼及在成魚的精巢和卵巢皆有 綠色螢光蛋白的表現。接著以連續刪除方式將PSR promoter 切割成不同大 小的片段,使用EGFP與Luciferase兩種報導基因的方式,發現在psr基因start cordon上游300 bps是啟動基因最重要的功能區域,並且推測repeat片段(約3 kb)可能具有enhancer的功能。本篇的研究發現PSR不只對於胚胎發育時期 重要,而且在後期骨骼與生殖腺的發育有重要關係。 關鍵字 : 斑馬魚、綠螢光蛋白、生殖腺、骨骼、磷脂醯絲胺酸受器、起動 子、基因轉殖 Abstract the The general function of phosphatidylserine receptor that PSR can recognize apoptotic bodies for clearance of corpse cells, but they temporally and spatially expressive pattern is still unclear. In this thesis, we cloned and constructed the PSR promoter that fused with green fluoresce protein for directly monitoring the temporal and spatial profile of PSR expression during zebrafish embryogenesis. We demonstrated that the 4kb of upstream sequence of PSR gene, which contain promoter region that can drive report gene EGFP expression in early zebrafish embryos. In the PSRP-4K-EGFP transgenic zebrafish fish line (F1), EGFP was expressed from oocyte to hatch out stages, especially, apparently expressed in somites, brain, eyes, heart, hatching gland and open of urinary duct. Interestingly, we first found that the PSR gene can express in skeleton (at the 14 dpf larvae) and testis or ovary (at adult stages). Furthermore, we constructed the different fragments of PSR promoter for the promoter activity assay. In the luciferase reporter assay, we found that a minimal requirement for PSR promoter activity is located in upstream 300 bps of start codon, and in the 3 kbps of upstream sequence that contain repetitive sequence may exist the enhancer elements. In this study, we discovered the PSR is not only important in embryogenesis, but also involved in skeletogenesis and gonadogenesis in late development stages. Keywords,EGFP, skeleton, gonad, promoter, PSR, transgenic, zebrafish