MOLECULAR CLONING AND DEVELOPMENTAL EXPRESSION OF AROMATASES GENE IN TWO SPECIES OF LOACHES

Fishes serve as a key link on the view of the vertebrate systematic evolution.Its sex determination is primitive and plastically compared with the other advanced vertebrates.Sexual differentiation of gonad is sensitive to environmental factors during a critical period of embryogenesis in some fish.The cytochrome P450 aromatase, which converts androgens to estrogens, is a key enzyme in sex differentiation of gonad.Therefore, the cytochrome P450 aromatase(CYP19)genes control the pathway of gonadal sex differentiation radically.Misgurnus auguillicaudatus and Paramisgurnus dabryanus are common Cypriniformes fishes in China.They have a very close relationship and were classified to the same genus by some taxonomists.Heteromorphic sex chromosomes have been found only in P.dabryanus.To detect the effect of temperature to sex differentiation, Molecular cloning and developmental expression of aromatases gene in two species of loaches were studied.The zygotes of Misgurnus anguillicaudatus and Paramisgurnus dabryanus were incubated separately at 20 ℃, 25℃and 30℃, until that the sex could be distinguished.The results show that high temperature(25-30℃)can promote male-biased sex ration in both species loach.And with the temperature rising, the male sex rate gets higher.A pair of nested degenerate consensus primes was designed basing on the sequence of cytochrome P450 aromatase gene(CYP19b).Using nest-PCR method, the part genomic sequences of CYP19b in two species loach were amplified and cloned from genomic DNA.The DNA fragments of MaCYP19b and PdCYP19b are 1337bp and 1473bp respectively.The gene-specific primers were designed based on the genomic DNA sequence.Then using RT-PCR method, the corresponding cDNA of CYP19b in both species loach were amplified and cloned from total RNA that was isolated from ovary tissue.By comparing their genomic DNA sequence and cDNA sequence, we know that the cloned CYP19b of two species loach all include 3 introns and 4 extrons and encode 145 amino acids.With the control gene of GAPDH, the expression patterns of the two genes were studied in adult tissues and different stages of embryo of two species loach separately with semi-quantitive RT-PCR. The results showed that M.anguillicaudatus CYP19b gene expressed only in ovary and kidney of adult tissues and gastrulae and neurula of embryo.The P.dabsryanu CYP19b expressed in brain, ovary and kidney of adult tissues and neurula and yolk-sac absorption phase of embryo.Those results lay the foundations of clarifying the relation between cytochrome P450 aromatase gene and the mechanism of ESD to a certain extent.