利用基因轉殖魚探討鯉魚(Cyprinus carpio)視紫質基因的調控機制

Abstract

視紫質(rhodopsin)是位於視網膜視桿細胞(rod cell)中的一種受光蛋白。目前在水生生物方面，並無視紫質基因調控機制方面的研究。為了瞭解鯉魚(Cyprinus carpio)視紫質基因其分子調控的詳細機制，利用由不同長度上游序列、以及突變某些鹼基的啟動子(promoter)來驅動水母綠螢光蛋白(EGFP)基因，並檢視其在轉殖魚的表現狀態。結果顯示，啟動子-6 kb?+74 bp，-1.2 kb?+94 bp，-138 bp?+94 bp，以及-76 bp?+94 bp的DNA片段皆能驅使轉殖基因在視網膜表現綠色螢光，而以-58 bp?+94 bp為啟動子的DNA片段則否。這代表-76 bp?+94 bp序列之中含有促使視紫質基因在視網膜表現的必要元件。在鯉魚視紫質基因上游-76 bp序列之內，包含了可能和轉錄因數Nrl及Crx結合的cis-elements。若將-76 bp?+94 bp啟動子中的-73 bp?-68 bp (putative Nrl response element)加以突變或是將其中-52 bp?-46 bp之間的序列(putative Crx binding site)移除，皆會致使此啟動子失去驅動轉殖基因在視網膜表現的能力。故此二cis-elements的共同存在對於鯉魚視紫質基因在視網膜中的表現是十分重要的。另外，注射以-6 kb?+74 bp和-1.2 kb?+94 bp為啟動子的轉殖魚，其綠色螢光在視網膜的表現率分別為62.3%及69.2%；相對之下，注射以-138 bp?+94 bp以及-76 bp?+94 bp為啟動子的轉殖魚，其表現率則分別為4.3%及3.6%。所以推測在-1.2 kb?-138 bp之間可能有強化子(enhancer)的存在。

Rhodopsin is one of the photoreceptor proteins, which locates at rod photoreceptors in the retina. Studying on the regulatory mechanisms of rhodopsin gene in aquatics is extremely limited. To further characterize the regulatory mechanisms of carp (Cyprinus carpio) rhodopsin gene, transgenic medaka (Oryzias latipes) carrying various deletions and mutations of rhodopsin gene promoters fused with enhanced green fluorescence protein (EGFP) cDNA were examined. The results showed that promoters ranged from -6 kb to +74 bp, from -1.2 kb to +94 bp, from -138 bp to +94 bp, and from -76 bp to +94 bp were able to direct retinal expression, whereas promoter ranged from -58 bp to +94 bp was not. This suggests that upstream sequence from -76 bp to +94 bp contains elements required for retinal expression. Within the upstream -76 bp sequence, there exist putative binding sites for transcription factors Nrl and Crx. The -76 bp to +94 bp promoter with mutations in -73 bp to -68 bp sequence (putative Nrl response element) failed to drive retinal expression. The same result was obtained by removing the -52 bp to -46 bp sequence (putative Crx binding site) from the -76 bp to +94 bp promoter. Thus, both of these two cis-elements were essential for rhodopsin gene expression in retina. Moreover, the rates of green fluorescence appeared in the eyes among transgenic fish were 62.3% and 69.2% for embryos injected with -6 kb to +74 bp and -1.2 kb to +94 bp fragments, respectively. In contrast, the eye-expression rates were 4.3% and 3.6% for embryos injected with -1.2 kb to +94 bp and -138 bp tp +94 bp fragments, respectively. Therefore, there may exist enhancer(s) of rhodopsin gene within upstream sequence from -1.2 kb to -138 bp.