Molecular Cloning Of CYP19 Gene And Their Expression Of Adult And Embryo In Two Species Of Loaches

Cytochrome P450 aromatase can be abbreviated as P450arom or CYP19. The enzyme aromatase P450(P450 arom) catalyzes the conversion of androgen to oestrogen and plays the role of maintaining physiological balance between sex steroid hormones. The study of the CYP19 gene is important to understand the molecular of sex differentiation and sex determination in fish.Four part genomic sequences of CYP19 gene were derived from two species of loaches brain and ovary, using RT-PCR and RACE. Through similarity searching in GenBank, two of these sequences contain the most similarity with DrCYP19a(83%), the rest two sequences contain the most similarity with GrCYP19b(84%). In terms of general nomenclature rule, they are named MaCYP19a,PdCYP19a and MaCYP19b,PdCYP19b . The CYP19a gene fragments of M.anguillicaudatus and P.dabryanus are as long as 1815bp and 1802bp, with 503bp 3'UTR and 506 3'UTR , which encodes 432 and 430 amino acids respectively. The CYP19b gene fragments of two species of loaches are as long as 428bp and 429bp, both encoding 142 amino acids. Phylogenetic analysis of the CYP19 gene by using MEGA4.0 indicated the CYP19a gene in the two species of loaches is clustered with other fishes ovary-derived P450arom gene, and is the closest with Danio rerio. And the CYP19b gene in the two species of loaches is clustered with other fishes braid-derived P450arom gene, and is the closest with Cyprinus carpio.With the reference gene of GAPDH, the expression of these genes in adult tissues and different stages of developing embryo of two species loaches were studied separately with the method of semi-quantitive RT-PCR and the fluorescent real time quantity RT-PCR. The results showed the two types of the CYP19 gene are highly expressed in brain and gonad, but significant difference between CYP19a and CYP19 b gene in embryo.