黑鯛卵黃前質基因 cDNA 部份序列之分析

Abstract

黑鯛(Acanthopagrus schlegeli)是原雄性雌雄同體魚種(Protandrous hermaphrodite)，小於一歲時為具有兩性生殖巢之個體，一足歲時達到雄性成熟(Functional male)，此後一直到二至四歲時才性轉變為雌性個體(Functional female)。利用反轉錄聚合酵素鏈反應(RT-PCR)的方法，選殖到四段黑鯛卵黃前質基因的部份cDNA，互補引子的設計來自二個方面，一是根據已經發表的脊椎動物卵黃前質氨基酸序列進行比對，找出相似的五段區域，設計六條互補引子，二是依所得到的RT-PCR產物BP-EI和BP-BE，定出其核酸序列，依序再設計了二條互補引子。 黑鯛卵黃前質基因cDNA的部份序列已定序出1750bp，這是目前第一篇關於雌雄同體魚種的卵黃前質cDNA被定序出來，經北方轉印和雜交試驗估計mRNA全長約6kbp，而且卵黃前質mRNA可以在經雌二醇誘導的雌魚和八個月大的未成熟幼魚的肝臟中被偵測到。轉譯出來的583個氣基酸序列和其他已經發現的脊椎動物的卵黃前質基因比較，之間有40-68％的相似，對於Glycosylation的位置和一些影響蛋白質二次結構之氨基酸(Cys, Trp, Tyr, Phe, Pro)所在的位置，在卵黃前質基因結構上都呈現高度的保守性。

The black porgy, Acanthopagrus schlegeli, is a portandrous hermaphroditic teleost. They become functional male at one-year- old, and eventually change their sex to be functional female between two and four years old. A reverse transcription polymerase chain reaction (RT-PCR)-assisted approach was used to isolate four partial vitellogenin (Vtg) cDNA clones, complementary to mRNA synthesized in the liver of estradiol-treated black porgy. The primers were designed by referring papers and data. The four partial Vtg cDNAs were sequenced and overlapped to be a 1,750 bp long cDNA. This is the first report about Vtg gene sequence in hemaphroditic teleost to be analyzed. The Vtg mRNA is estimated about 6 kbp by northern blot and hybridization. The Vtg mRNA was detected in livers of both mature female fish and estradiol-treated 8-months-old juvenile fish. The primary structure of the deduced 583-amino-acid protein is 40~60% identical to other documented vertebrate Vtgs. The possible glycosylation sites and some amino acids (Cys, Trp, Tyr, Phe and Pro) are highly conserved. This results indicate that the secondary structure of Vtg may be conserved in veterbrates.