条斑星鲽CYP19a基因克隆及其在雄鱼生殖周期中的表达

Cloning of CYP19a gene and expression analysis during the reproductive cycle in males of Verasper moseri

摘要:

细胞色素P450芳香化酶(P450arom)作为P450细胞色素酶超家族中的一员，是性类固醇生成途径中的末端酶，能够将雄激素转化为雌激素。在大多数脊椎动物中，P450arom由CYP19单基因编码。但是在鱼类中存在两种P450芳香化酶，分为性腺型芳香化酶(P450aromA)和脑型芳香化酶(P450aromB)，它们由不同的基因(CYP19a和CYP19b)编码，分布在不同的组织。通过简并引物扩增及RACE cDNA扩增克隆，在国内外首次获得全长为2 167 bp的条斑星鲽CYP19a cDNA序列，并将推测的氨基酸序列与其它物种P450arom氨基酸序列进行多重比较，发现存在跨膜螺旋区、I-螺旋区、Ozol肽区、芳香化酶特异保守区以及血红素结合区。通过RT-PCR分析了P450aromA mRNA在条斑星鲽不同组织中的表达情况，结果表明,CYP19a基因主要在脑、卵巢和精巢中表达，其次在肠、肝脏、肾脏也有少量表达。同时也分析了P450aromA mRNA在处于不同发育期的精巢中的表达情况，发现在Ⅱ期精巢中表达量最高，在Ⅴ期精巢中表达量最低。

Abstract:

Estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase is the terminal enzyme in steroidogenic pathway that converts androgens into estrogens. In most of vertebrates, P450arom is the product of the CYP19 gene, which exists as a single copy per haploid genome. In contrast, there are two isoforms of aromatase in teleosts, which are encoded by two different genes, CYP19a and CYP19b, as well as have different distribution. This work describes the molecular cloning of the cDNA encoding the barfin flounder (Verasper moseri) gonad cytochrome P450 aromatase(CYP19a) by means of reverse transcriptase polymerase chain reaction(RT-PCR), degenerate primers PCR amplification and 5′ and 3′rapid amplification of cDNA ends(RACE) analyses. We cloned the complete cDNA sequence of CYP19a gene and the length is 2 167 bp, encoding a protein of 523 amino acids. Via Blastn, the CYP19a cDNA displayed high identity with Atlantic halibut (Hippoglossus hippoglossus), Japanese flounder (Paralichthys olivaceus) and southern flounder (Paralichthys lethostigma). Alignment analysis using barfin flounder CYP19a gene deduced animo acid sequence and animo acid sequences of other species showed that there were transmembrane helix region, I-helix region, Ozol’s peptide region, aromatasespecific conserved region and hemebinding region in the deduced animo acid sequence. Phylogenetic analysis suggested the occurrence of a gene duplication for cytochrome P450 aromatase in the teleost lineage after its divergence from the tetrapods. Furthermore, the higher identity between barfin flounder P450aromA and P450aromAs of other teleosts suggested that they have the same origin. A semiquantitative PCR was developed to measure mRNA expression levels of CYP19a gene of barfin flounder. Expression analysis in different tissues demonstrated that P450aromA transcripts were highly abundant in brain, ovary and testis, less abundant in intestine, liver and kidney, showing that not only gonads can produce steroids but also other tissues have steroidogenic capability. And we analyzed the relative mRNA expression level of CYP19a in testes at different stages of reproductive cycle, there were the highest level at stageⅡ and the lowest level at stageⅤ. These changes in expression profiles may imply differences in the functionality of the enzyme between different stages, suggesting an important role for P450aromA in barfin flounder testes development.