斑馬魚肌肉調控蛋白myf-5基因其內含子序列抑制體節專一表現的新發現

Abstract

Myf-5 是肌肉特異性轉錄因數之一，參與調控肌原纖維及肌肉細胞的增生和分化。目前對於調控myf-5基因的cis-acting element並不十分清楚，故本實驗利用基因轉殖的技術，以斑馬魚(Danio rerio)作為實驗材料，研究參與調控myf-5基因組織及時期專一表現的區域。在構築質體中，其中包含斑馬魚myf-5上游調控區-9977至-1(-9977/-1 ) , exon 1 (E1) , intron 1 (I1) , exon 2 (E2) 再in frame結合GFP cDNA [(-9977/-1)/El/I1/E2/GFP]，以顯微注射方式將其注射至單細胞時期的斑馬魚胚胎中。結果顯示螢光在體節專一表現率僅 2％；相對地，在不含有E1/I1/E2序列之質體(-9977/-1)/GFP螢光表現率卻有94％。相同的顯微注射結果也被觀察到在-8600/-1，-2937/-1 和-290/-l 結合El/Il/E2/GFP 的實驗中。進一步地運用連續性剔除造成不同長度的intron片段，構築質體-2937/-1/GFP 連接 intron 1片段+502/+2503，+1174/+2503，+1768/+2503，+790/+1489，+1467/+2152，+502/+1787，+502/+1199，+502/+835和+502/+659，經顯微注射發現，轉殖胚胎體節專一螢光表現率分別為2，90，88，87，83，12，10， 8及83％(質體-2937/-1/GFP螢光表現率為84％)，表示intron 1中的+659/+835序列有能力抑制螢光載體節專一的表現。並且在剔除質體(-2937/-1) /GFP/(+502/+1199)中之+660/+816序列，注射後體節專一螢光表現率即會上升至91％。由上述證據顯示，intron 1的+660/+816序列抑制了myf-5上游調控區的表現，而且發現其抑制能力是具有位置及方向的特異性。因此，myf-5 intron 1 內+660/+816 序列，可能在斑馬魚myf-5組織特異性及發育時期特異性表現上扮演重要角色

Myf-5 is the one of the muscle regulatory factors involved in the proliferation of myoblasts and differentiation of myogenic cells. Yet, the cis-regulatory elements of myf-5 gene are not clearly defined. Due to no particularly suitable cell-lines available for analyzing the gene regulation of zebrafish myf-5, in vivo transgenic assay was employed. A plasmid which contained zebrafish myf-5 gene upstream regulatory sequence from -9977 to-1 (-9977/-1), exon 1 (E1), intron 1 (I1), exon 2 (E2) and in frame fused with GFP [(-9977/-1)/E1/I1/E2/GFP], was constructed and microinjected into one-cell embryos. Surprisingly, the somite-specific expression rate was extremely low (2％, n=392), compared to that (94％) of embryos injected with (-9977/-1)/GFP. Similar results were also obtained by injecting the constructs of E1/I1/E2/GFP but fused with -7710/-1, -2937/-1 and -290/-1. To further study the regulatory elements within the intron, various deletion fragments of intron 1 were finely dissected and fused with upstream -2937/-1 and GFP [(-2937/-1)/GFP]. The somite-specific expression rates were 2, 90, 88, 87, 83, 12, 10, 8 and 83％ for embryos injected with (-2937/-1)/GFP fused with intron 1 +502/+2503, +1174/+2503, +1768/+2503, +790/+1489, +1467/+2152, +502/+1787, +502/+1199, +502/+835 and +502/+659, respectively. Whereas, the somite-specific expression rate of embryos injected with (-2937/-1)/GFP was 84％, which was quite close (91％) to that of embryos injected with fragment (-2937/-1)/GFP/(+502/+1199) but absence of +660/+816 segment. These evidences strongly suggest that downstream sequence of +660/+816 motif represses the expression of zebrafish myf-5 gene. This is the first report to propose that an intron segment may be involved in delicately controlling the somite-specific and stage-dependent expression of myf-5.