黄颡鱼卵巢P-450arom基因的克隆及组织表达

Molecular cloning of ovarian P450arom gene and its expression in male and female yellow catfish 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.本研究采用RT-PCR和RACE(Rapid Amplification of cDNAEnds)法,分离和克隆了黄颡鱼(Pelteobasrus fulvidraco)卵巢P450芳香化酶基因HP450arom A,并使用荧光实时定量RT-PCR对其组织表达进行了分析.结果表明,HP450arom A cDNA全长1914 bp[不包括poly(A)],5′端非翻译区有13 bp,3′端362 bp[不包含poly(A)],阅读框(Open Reading Frame,ORF)1 539bp,翻译成513个氨基酸,计算的蛋白质分子量为58.7 kD.同源性分析显示,HP450arom A的氨基酸序列与其他鱼卵巢P450arom具有60%～90%的同源性,与其他鱼脑P450arom为57%～60%同源,与鸡卵巢和人胚盘P450arom则为51%和52%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和血红素结合区Ⅲ和其他鱼芳香化酶相比同源性分别高达68%～97%、78%～91%和71%～100%.系统发育分析表明,HP450arom A与鱼类卵巢P450arom属于同一分支,黄颡鱼和鲇鱼亲缘关系最近,这和传统的分类方法结论一致.荧光实时定量RT-PCR研究结果显示,HP450arom A在前脑、下丘脑、脑垂体、精巢、肝脏中不表达,只在卵巢中表达.这说明HP450arom A的表达具有组织特异性,并可推测其对卵巢发育起着重要作用.与本室分离的HP450arom B在卵巢的表达量相比,卵巢中HP450arom A的表达量是HP450arom B的18.7倍.

The enzyme aromatase P-450（P450arom）can catalyse the conversion of androgen to oestrogen. A cDNA encoding P450arom was derived from yellow catfish （ Pelteobagrus fulvidraco ） ovary using RT - PCR and RACE. The cDNA was 1 914 bp with 13 bp 5＇UTR, 362 bp 3＇UTR[excluding poly（A）] and 1 539 bp ORF, which encoded 513 amino acids and had a predicted mol wt of 57.8 kD. The yellow catfish ovarian P450arom shared 60 % - 90 % sequence identity with ovarian aromatases of other four fish species, which were golden fish, channel catfish, rice eel and zebra fish, but shared only 57 % - 60 % with brain-derived aromatases of the four fishes, 51% and 52% with chicken ovarian and human placenta 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 heine-binding region Ⅲ, which were 67 % -96 %, 78% -86% and 78% - 100% respectively. Phylogenetic analysis of the P450arom gene family indicated the yellow catfish ovarian P450arom was clustered with the four fishes＇ ovarian P450arom, and yellow catfish was most close with channel catfish . This result is consistent with that of contradiction classification. The fluorescent real-time quantity RT-PCR analysis reveals that HP450arom A expresses only in ovary, but not in yellow catfish brain, liver nor testis. The comparison of HP450arom A and HP450arom B expressions in ovary indicates the expression level of HP450arom A is 18.7 times of that of HP450arom B.