黄鳝P-450芳香化酶基因的克隆及序列分析

CLONING AND SEQUENCE ANALYSIS OF THE cDNA ENCODING P-450 AROMATASE FROM RICE FIELD EEL

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(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。

In both males and females, estrogen programs and coordinates developmental, physiological, and behavioral responses essential for reproduction. The cyp19 gene encodes P450arom, a heme-binding protein of the enzyme complex responsible for the conversion of Cl9 androgens into Cts estrogens. This enzyme complex comprising the flavoprotein NADPH-dependent cytochrome P450 reductase and P450 arom. Studies show that aromatase may influence the function and development of Central Nervous System of the mammals, readjust nervous internal secretion, reproduction function and sexual behavior, and participate the readjustment of sexual gland differentiation of non-mammals. Many experiments have proven aromatase can control the sex differentiation and sex transformation of many kinds of fishes. Rice field eel （ Monopterus albus） is a kind of protogynous hermaphrodite. The studies of molecular mechanism of the rice field eel sex determination are not available. This study isolates P450arom gene from ovary of rice field eel. It intends to further study the function of P450arom during sex formation and sex reversal as well as the expression levels of the genes after the application of hormone and aromatase inhibitor in the future, aiming to provide basic materials for artificial sex control of rice field eel. A cDNA encoding P450arom was derived from the rice field eel ovary using RT-PCR and RACE. The cDNA was 1802bp with 49bp 5＇UTR,202bp 3＇UTR（excluding poly（A）） and 1551bp ORF, which encodes 517 amino acids and has a predicted mol wt of 58.2K. Based on mutational analysis and molecular modeling amino acids known to be essential for catalytic functions in the human P450arom（1133, E302, P308, D309, T310, R435, C437） were identical in rice field eel P450arom. Consistent with the glycosylation site described at the N terminus of human aromatase, a consensus N-glycosylation site （N-X-S/T） was identified in the amino terminal region of rice field eel at N29, which is similar to goldfish ovarian P450arom （N30）. Further analysis indicated that the rice field eel P450arom has protein kinase C （PKC）-dependent sites at 59,130,385,410, and 503 （S/T-X-R/K）. For phylogenetic analysis the deduced amino acid sequence of rice field eel, together with P450arom sequences reported for other vertebrate species, were aligned by ClustalW, version 1.6. The results revealed the rice field eel ovarian P450arom shares 63 %--80 % sequence identity with ovarian aromatases of other fish species, but only 58 %--60 % with brain-derived aromatases of other fishes,50%--52% with human being placenta and chicken ovarian aromatases. But the percent of identity/similarity was higher （70%--100%） in the regions of high homology, including the l-helix, an aromatase-specific conserved region, and the heme-binding region. The aligned sequences were used to construct phylogenetic trees by distance （Neighbor-Joining algorithm） and maximum parsimony criteria PAUP （Phylogenetic analysis using parsimony, version 4.0）. Phylogenetic analysis of the P450arom gene family indicated that P-450 aromatase gene has a single origin. The rice field ovarian P450arom was clustered with medaka ovarian P450arom, and all fish ovarian aromatase were clustered together, and is independent with fish neural P450arom and chicken human P450arom.