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P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE法,首次分离和克隆了黄颡鱼脑P450芳香化酶基因HP450aromB,并使用荧光实时定量RT-PCR对其组织表达进行了研究。结果表明HP450aromB cDNA全长2080 bp(不包括poly(A)),5′端非翻译区有25 bp,3′端555 bp(不包含poly(A)),阅读框(open reading frame,ORF)1500 bp,编码500个氨基酸,推测的蛋白质分子量为56 kDa。同源性分析显示,HP450aromB的氨基酸序列与其它鱼脑P450arom具有70%以上的同源性,与其它鱼卵巢P450arom为60%左右同源,与人胚盘和鸡卵巢P450arom则为50%左右同源;但芳香化酶高保守区包括I-螺旋区,芳香化酶特异保守区II及血红素结合区III和其它鱼芳香化酶相比同源性分别为83%~96%,78%~86%和85%~100%。系统发育分析表明HP450aromB与鱼类脑P450arom属于同一分支的,并且也显示了黄颡鱼和鲶鱼分类地位最近,这和传统的分类一致。实时定量RT-PCR研究显示,HP450arom B在前脑,下丘脑,垂体、卵巢、精巢均有表达,在肝脏没表达,表达量脑部高于性腺,但雌雄鱼脑部HP450arom B的表达总量没显著差异。
P-450 aromatase (P450arom) is a key enzyme that catalyzes the biosynthesis of estrogen by androgens. In this paper, the aorta P450 aromatase gene HP450aromB was isolated and cloned for the first time by RT-PCR and RACE, and its tissue expression was studied by real-time quantitative RT-PCR. The results showed that the full-length cDNA of HP450aromB was 2080 bp in length (excluding poly (A)), 25 bp in 5 ’untranslated region, 555 bp in 3’ end (without poly (A)), open reading frame (ORF) 1500 bp, encoding 500 amino acids, suggesting a molecular weight of 56 kDa. Homology analysis showed that the amino acid sequence of HP450aromB shared more than 70% homology with other fish brain P450arom, which was about 60% homologous to P450arom in other fish ovaries and about 50% with P450arom in human embryo disk and chicken ovary However, the homology of the aromatase high conserved region including the I-helix region, aromatase-specific conserved region II and heme-binding region III was 83% -96%, 78% ~ 86% and 85% to 100%. Phylogenetic analyzes showed that HP450aromB belonged to the same branch as fish brain P450arom and also showed the most recent taxonomic status for yellow catfish and catfish, consistent with the traditional classification. Real-time quantitative RT-PCR showed that HP450arom B was expressed in the forebrain, hypothalamus, pituitary, ovary and testes, but not in the liver. The expression of HP450arom B was higher in the brain than in the gonad, but the total amount of HP450arom B No significant difference.