黄颡鱼脑P-450芳香化酶基因的克隆和组织表达

Molecular cloning of neural P450arom and its expression in Pelteobagrus fulvidraco

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.

The enzyme aromatase P-450（P450arom） catalyzes the conversion of androgen to oestrogen. A cDNA encoding P450arom was derived from yellow catfish （Pelteobagrus fulvidraco） brain, using RT-PCR and RACE. The cDNA was 2080 bp with 25 bp 5′ UTR, 555 bp 3′ UTR（ excluding poly（A） ） and 1500 bp ORF, which encodes 500 amino acids and has a predicted mol wt of,56 kDa. The yellow catfish brain-derived P450arom shares above 70% sequence identity with brain-derived aromatases of other fish species, but only about 60% with ovarian aromatases of other fishes, about 50% with human placenta and chicken ovarian aromatases. But the percentage of identity/similarity was higher in the regions of high homology, including the Ⅰ-helix, an aromatase-specific conserved region Ⅱ, and the heme-binding region Ⅲ, their homology being 823% - 96%, 78% - 86% and 85% - 100% respectively. Phylogenetic analysis of the P4.50arom gene family indicated the yellow catfish brain-derived P450arom was clustered with other fishes brain-derived P450arom, and is the closest with channel catfish. This result is consistent with the result of traditional classification. The fluorescent real time quantity RT-PCR analysis revealed HP450arom B is expressed in fore-brain , hypothermia, pituitary, ovary and testis, but is high in brain and low in gonad. There is no expression in liver. There is no significant difference between male fish and female fish brain.