赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,

Cloning and expression of two cytochrome P450 aromatase genes in red-spotted grouper Epinephelus akaara

The cDNA sequences encoding two distinct cytochrome P450 Aromatases, namely P450aromB and P450aromA, were isolated from pituitary of the red-spotted grouper Epinephelus akaara by GeneRacer~(TM). The P450aromA cDNA is 1 833 bp in length, and the open reading frame encodes a putative protein of 518 amino acids. The P450aromB cDNA is 1 901 bp in length, and the open reading frame encodes a putative protein of 509 amino acids. Sequence and phylogenetic analysis showed that these two P450arom genes of the red-spotted grouper are the orthologs of previously identified P450arom genes in the orange-spotted grouper, and results were similar to those analyzed by traditional morphology and biochemistry in their evolution status. The sequence analysis indicated that the cDNA sequence and the amino acids homogeneity between them were 51.6% and 62.5%, respectively, and the highest identity to orange-spotted grouper were 94.7% (P450aromB) and 97.9%(P450aromA). The expression of both P450aromA and P450aromB genes in different tissues of the red-spotted grouper were further examined using RT-PCR in 21 tissues between male and female fish. The expression in gonad(P450aromA > P450aromB) was opposite to that in brain(P450aromB > P450aromA), there were overlapping expression of the two genes in neural and gonadal tissues .