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

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.