蔡懷楨臺灣大學：分子與細胞生物學研究所劉家宏Liu, Chia-HungChia-HungLiu2007-11-252018-07-062007-11-252018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/49926摘要： 許多基因之mRNA的5’端不轉譯區中被證明有upstream open reading frame（uORF）的存在，而且會對其下游的基因產生轉譯上的抑制。這些含有uORF的基因都具有重要的功能並需要受到精密地調控。然而，目前關於uORF的研究都集中在非活體型態(in vitro)的實驗，以活體(in vivo)探討uORF功能的研究則甚為缺乏；另一方面，uORF的調控與發育之間的關係，也有許多待研究之處。本研究試以斑馬魚作為模式動物，探討人類Chop基因的uORF與發育間的關聯性。結果顯示，人類Chop基因的uORF可同樣在斑馬魚中產生作用；利用序列比對結果，發現各物種間的Chop基因mRNA皆含有uORF，彼此的序列也有相當的保守性。利用突變實驗結果發現，保守存在於各物種Chop基因uORF序列中的兩個AUG皆在uORF的抑制作用上具有影響性，但以第二個AUG的影響最大。關於uORF抑制作用的調控，利用溫度逆境與MO注射的實驗得知，Chop基因的uORF會在溫度造成發育異常以及MO破壞肌肉分化時降低抑制作用，證實Chop基因uORF的轉譯調控在發育過程中具有特定的作用。此外，以mRNA over-expression與TUNEL assay來探討Chop的表現對胚胎發現的影響，發現Chop基因具有多種功能，除會影響胚胎的體軸發育外，亦會造成細胞凋零的現象。從上述的結果顯示，利用斑馬魚在胚胎發育研究上的優勢來研究uORF在in vivo環境中扮演的角色以及其和發育間的關係實屬可行。Abstract： The upstream open reading frame (uORF), which has the inhibitory effect on the translation of the downstream gene, has been found in the 5’-untranslated region(5’-UTR) of many genes. Among the genes with uORF, most of them are responsible for crucial roles in the cell and need to be regulated precisely. However, the studies on uORF are still executed mainly in vitro. In addition, the roles of uORF on the development are not clear. To investigate the in vivo role of Chop uORF on the development, we try to use the zebrafish, an excellent animal model for development study, for our study. In this study, we found that the uORF of human Chop gene could also exhibit its inhibitory effect in the zebrafish. By comparing the cDNA sequences, we found that uORF are conserved among many species . As the results of mutation experiment revealed, the two AUGs conserved in the uORF sequence among species were both involved in the inhibitory effects of uORF. The inhibitory ability of Chop uORF was shown to be under regulation by the temperature stress and knockdown experiments, in which the translational inhibition exerted by uORF was reduced in abnormally-developing embryos, indicating that the Chop uORF might be involved in the development. In the mRNA overexpression experiment and TUNEL assay, the results suggested that Chop might play a role in the development by influencing axis formation and apoptosis. The results showed that zebrafish might be a feasible animal model for investigating the in vivo function of uORF and its role in the development.目錄 誌謝------------------------------------------------- 1 中文摘要--------------------------------------------- 5 英文摘要--------------------------------------------- 7 前言------------------------------------------------- 8 實驗材料與方法--------------------------------------- 14 結果------------------------------------------------- 21 討論------------------------------------------------- 28 參考資料--------------------------------------------- 35 圖表------------------------------------------------- 39 附錄------------------------------------------------- 522008795 bytesapplication/pdfen-US斑馬魚發育uORFzebrafishchopdevelopmentupstream open reading frameotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49926/1/ntu-96-R94b43032-1.pdf