The Cloning Of Cyp19a1a CDNAs And A Comparative Study Of Its Expression In Different Polyploidy Cyprinid Fishes

Cytochrome P450aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen.There are two isoforms of aromatase in teleosts, which are encoded by two different genes, cypl9ala and cypl9alb, as well as have different distribution.The duplicated copies of fish cypl9genes were evolved to the brain and ovarian forms of cytochrome P450aromatases respectively, namely cypl9ala and cypl9alb with different regulatory mechanisms of expression. The former one, which mainly expresses in ovary, takes parts in estrogen biosynthesis and ovary differentiation, and plays a leading role in differentiation of sex gland, while the latter one mainly expresses in brain. According to researchers, estrogen acts a vital role in sex differentiation, ovarian follicle development and female character maintenance. The estradiol in the estrogen, especially, acted as sex steroid, participates in many critical processes, including growth, development and breeding, etc.The formation of allotetraploid population derived from red crucian carp (carassius auratus red var.,♀,2n=100) x common carps (cyprinus carpio L.,♂,2n=100), is the first case artificial polyploid fish with the bisexual fertile and stable inheritance in vertebrate in the wold,which played an important role in biological evolution and fish selective breeding. The triploid crucian carp was produced by mating the males of allotetraploids with the females of Japanese crucian carp (Carassius auratus cuvieri), which have shown several enhanced performances such as sterility, faster growth rate, good flesh quality and higher anti-disease ability. Both tetraploids and triploids provided a biological platform for the comparative studies of different ploidy level fishes and for cypl9ala gene in polyploid fish function and mechanism research.All these results together suggest that cypl9ala gene plays a vital role in the process of germ-line development and closely linked with the sterility of allotriploid crucian carp.In this study, we firstly cloned the cDNAs and comparatively analyzed the expressions of cypl9ala gene in different ploidy fishes.The main topics in this paper were presented as follows:1) By RT-PCR and RACE, we obtained the cDNA sequences of cypl9ala of triploids and tetraploids. The cDNA length of allotetraploid hybrids is1960bps and encoding517amino acids; the cDNA allotriploid crucian carp is1782bps and encoding517amino acids. The female parent of allotriploid crucian carp and the allotetraploid hybrids original female parent red crucian carp cyp19ala full length cDNA sequence was1954bps, encoding517amino acids. Sequnece alignment and phyiogenetic analysis revealed that triploids and tetraploids cypl9ala putative proteins had higher amino acid similarities with the same proteins of other fish, while lower similarities with mammalian species, which confirmed their identities. These results indicted that the related genes of cypl9ala is conservative in evolution. RT-PCR analysis revealed that cypl9ala was detectable only in allotriploid crucian carp testes, ovaries, brain and spleen.2) We used the Real-time PCR to study the cyp19ala mRNA level in the ovary and testis of the different ploidy fishes. In spawning season, the expression in ovary of triploids was the lowest; the expression in testis of triploids was the highest. However, in prespawning season, the expression in ovary of triploids was the highest. The abnormal expression of cyp19ala in triploids during the breeding season may for the main function of cypl9ala is turning the androgen to estrogen.3) We used the western-blot to study the CYP19A protein level in the ovary and testis of the different ploidy fishes. In spawning and prespawning season, the expression in ovary of triploids was the lowest. The result precisely shows that the estrogen expresses insufficiently in triploids.