暗纹东方鲀芳香化酶基因的结构及表达分析

性腺型芳香化酶基因是一种调节雌雄激素平衡的基因。为初步探究暗纹东方鲀性腺型芳香化酶基因在性腺发育和性别分化过程中的作用,通过RACE及荧光定量PCR技术,成功克隆出暗纹东方鲀性腺型芳香化酶基因的cDNA全长序列,共1786 bp,编码514个氨基酸,与红鳍东方鲀及星点东方鲀同源性最高。氨基酸序列分析显示,该基因编码的蛋白是一种稳定的亲水性蛋白,但不存在信号肽序列;性腺型芳香化酶含有38个磷酸化位点、4个N-糖基化位点、2个跨膜保守结构域;其氨基酸序列在哺乳动物和鱼类中均十分保守,且含有跨膜区、Ⅰ-螺旋区、Ozol′s肽区、芳香化酶特异性保守区和亚铁血红素结合区等功能保守区。表达分析显示,暗纹东方鲀性腺型芳香化酶基因主要在肌肉和卵巢中表达,其次在其他组织中也有少量表达,而且在幼鱼不同发育期卵巢中的表达量呈现出逐渐升高再降低的表达趋势,在精巢中则基本不表达。研究结果表明,性腺型芳香化酶基因很可能参与了暗纹东方鲀雌鱼性腺分化后卵巢的发育、雌性特征的维持和其他组织的发育等过程。

Molecular Cloning,Sequence Structure and Expression Analysis of Gonadal Aromatase Gene in Puffer Takifugu obscures

The gonadal aromatase gene(cyp19 a)was a kind of gene that regulates the sex hormone balance.The purpose of this study was to preliminarily investigate the role of gonadal aromatase gene in gonad development and sex differentiation in puffer Takifugu obscurus,and we successfully cloned the full-length cDNA sequence of cyp19 a in puffer through RACE and Real-Time PCR technology.The full length of cyp19 a sequence was 1786 bp,encoding 514 amino acids,and sharing the maximal homology with the genes of puffer T.rubripes and T.niphobles.The amino acid sequence analysis showed that the protein encoded by cyp19 a was hydrophilic and stable protein,but there is no signal peptide sequence.There were 38 phosphorylation sites,4 N-glycosylation sites,2 transmembrane domains in the sequence.Meanwhile,the multiple amino acid sequence alignment showed that the amino acid sequence of gonadal aromatase was very conservative in both mammalian and fish,and contained some functional conservative regions such as the transmembrane region,I-helix region,Ozol′s peptide region,aromatase-specific conserved region and heme binding region.Furthermore,expression analysis showed that the cyp19 a was highly abundant in ovary and muscle,but less abundant in other tissues.The expression of cyp19 a in the ovary of juvenile fish at different developmental stages showed a trend of increasing first and then decreasing rapidly,without in the testis.The findings indicated that cyp19 a may be involved in the development of ovarian after gonad differentiation,maintenance of female characteristics and development of other tissues in female puffer.