摘要
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.
出处
《水产学报》
CAS
CSCD
北大核心
2005年第5期591-598,共8页
Journal of Fisheries of China
基金
国家重点基础研究"973"规划项目(2004CB117401)
无锡市自然科学基金(CK03001)
关键词
黄颡鱼
P450芳香化酶
cDNA末端快速扩增法
系统发育
组织表达
Pelteobagrus fulvidraco
P450 aromatase
rapid amplification of cDNA ends（RACE）
phylogenesis
expression in tissues
作者简介
徐跑（1963～），男，江苏，研究员，博士研究生，主要从事鱼类育种及生物技术研究。E-mail：Xup@ffrc．cn
通讯作者：夏德全．E-mail：xiadq@ffrc.cn
