臺灣博碩士論文加值系統

生物體內的類固醇荷爾蒙經過ㄧ連串的酵素代謝反應後，依其功能可區分成四大類，分別是：鹽類皮質素、醣類皮質素、性類固醇荷爾蒙與神經類固醇荷爾蒙，在類固醇荷爾蒙生成反應裡，CYP11A1(P450scc)與HSD3B則分別是控制整個過程的第一與第二個關鍵酵素。在斑馬魚裡，經由5’-RACE，發現cyp11a1的基因結構與哺乳類不同，轉錄起點後有兩個不轉譯的exon；另外，藉由原位雜交法發現cyp11a1與hsd3b RNA會在卵子發育過程中被生產與儲存，並隨著卵子受精後傳至胚胎，因此都屬於斑馬魚胚胎的母體因子；使用HSD3B酵素分析來偵測HSD3B蛋白的表現，發現HSD3B會出現在卵子周邊的濾泡細胞；但是在卵子與剛受精的早期胚胎裡，卻都沒有發現HSD3B蛋白；比較hsd3b RNA與HSD3B出現的時間與位置以後可以發現，hsd3b RNA與HSD3B蛋白在斑馬魚卵子發育過程與胚胎發育早期的表現並不相同，有hsd3b RNA的存在卻沒有蛋白的轉譯，因此可以知道在這段期間，hsd3b RNA發生了轉譯控制，使得蛋白無法被轉譯出來。最後利用在斑馬魚胚胎裡，打入帶有不同3’-UTR的GFP RNA來研究不同3’-UTR對GFP轉譯的影響，發現GFP報導基因的表現時間會受到所帶3’-UTR的影響，帶有hsd3b 3’-UTR的GFP和其他的控制組比較起來，的確有轉譯被抑制延後的現象；另外構築帶有cyp11a1與hsd3b mRNA 3’-UTR不同片段的報導基因，發現cyp11a1 3’-UTR 1~231 bp與hsd3b 3’-UTR 20~62 bp這段區域，可以調控GFP報導基因與帶有全長之報導基因在相同的時間開始表現螢光，因此可能有重要的轉譯調控序列坐落在這些序列上。

The major steroid hormones include minercorticoid, glucocoticoid, sex steroids and Neurosteroids. They control salt and water balance, glucose metabolism, and sexual characteristics. CYP11A1(P450scc) and HSD3B are first two enzymes involved in steroidogenesis. Using 5’-RACE, we found that zebrafish cyp11a1 have two untranslated exons. In zebrafish ovary, the transcripts of those two genes are located in the oocytes and granulosa/theca cells. Moreover, these transcripts can be detected as early as in 1-cell stage embryos. It indicates that the transcripts of those two genes are maternal mRNA. To further confirm, we used HSD3B activity assay to detect the protein location. Interestingly, HSD3B protein activity was only detected in ovarian granulosa/theca cells, but not in the oocytes and early stages embryos. These results suggested that the maternal hsd3b transcripts are under translational controls in oocytes and early stages embryos which are important for embryonic development. To determine whether the 3’UTR of cyp11a1 and hsd3b are involved in their translational control, GFP reporters linked to variant 3’UTR were constructed and their in vitro transcripts were injected into zebrafish embryos. We found that the transcripts with hsd3b 3’UTR expressed GFP later than that with cyp11a1 and globin 3’UTR. The results suggested that hsd3b 3’UTR may have machinery of translational control.

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