Cloning,Expression Of ERs,AR Genes In Cyprinus Carpio Var Singuonensis And The Effect Of EE2,MT On Their Expression In Liver Of Juveniles

Both environmental estrogens and androgens can disrupt normal endocrine function of wild animals and human, and they have similar effects on estrogen and androgen. EE2 and MT in the environment, as a kind of estrogens and androgen respectively, can disrupt normal endocrine function through interacting with estrogen receptors(ERs).The full-length cDNAs of estrogen receptors( ERα,ERβ,ERβ1, ERβ2), androgen receptor(AR) and part-length cDNAs of vitellogenin(vtg) were isolated and characteized from the Xingguo red carp(Cyprinus carpio var singuonensis). Full length cDNAs of 2109 bp ERα, consist of 189 bp 5’ Untranslated Regions(UTR), 237 bp3’UTR, 1683 bp Opening reading frame(ORF) translates 560 amino acids. ERβ is 2147 bp, consists of a 330 bp5’UTR, a 137 bp 3’UTR and a 1680 bp ORF which translates 559 amino acids. Full length cDNAs of 2300 bp ERβ1 consists of a 364 bp 5’UTR, a 178 bp 3’UTR, a 1758 bp ORF translates 585 amino acids. The full length of ERβ2 is 2007 bp, consists of a 156 bp 5’UTR, a 216 bp 3’UTR, 1635 bp ORF, the ORF translates 544 amino acids. The full length of 3229 bp AR consists of a 104 bp 5’UTR, a 485 bp 3’UTR, a 2540 bp ORF which translates 846 amino acids. Multiple alignments were performed using the Megalign program of Lasergene, the complete sequences of C. carpio var singuonensis ERs share high amino acid identities with their counterparts in cyprinid fish such as: Cyprinus carpio, Carassius auratus var. pengze,Onychostoma barbatulum,Gobiocypris rarus and Carassius auratus gibelio.Phylogenetic tree was constructed from amino acid alignment of ERs in C. carpio var singuonensis and other vertebrates using the Neighbor-Joining method, the phylogenetic analysis showed a clear separation of ERα and ERβ, ERβ1, ERβ2. Four subtypes of the ERs are groupd most closely with their counterparts of cyprinid fish. Phylogenetic analyze of C. carpio var singuonensis and other vertebrates AR showed that C. carpio var singuonensis AR are grouped closely with Carassius auratus gibelio, Carassius auratus and Carassius auratus var. pengze, but separated with Homo sapiens and Rattus norvegicus.Differential ERs and AR mRNAs expression levels were detected in tissues of one-year-old mature C. carpio var singuonensis by Quantitative real-time PCR(qRT-PCR). The ERs showed high mRNA expression in the liver, ovary and intestines of female, in male, ERα and ERβ mRNAs were mainly expressed in liver, with ERβ1and ERβ2 mainly transcribed in intestine and testis respectively. AR was mainly expressed in liver, ovary, kidney, muscle and cerebrum, with the highest expression level in liver. The mRNA expression of vtg was higher in liver, cerebrum and gill. ERs and AR mRNA transcription levels in C. carpio var singuonensis(150 dph) following EE2(0.01, 0.1 and 1 nmol/L) expose were detected by qRT-PCR. The ERα mRNAs was significantly up-regulated in the three groups treated by EE2 for 1 and 2 weeks. The ERβ mRNA expression was dramatically increased under EE2 exposure in our study. ERβ1 mRNA expression was suppressed by EE2 for 1 and 2 weeks. The expression of ERβ2 mRNA was suppressed in the 1-week group, with the highest suppression occurred at 0.01nmol/L of EE2. So the expression manner of four ERs genes in liver were different in exposed groups. In female, AR and vtg transcripts were detected in tissues of adult female fish. After expose to 50 μg/L MT(17α-methyltestosterone) for 4 weeks, The AR expression was weakly down-regulated in the first two weeks and was weakly up-regulated in the 3 and 4 weeks, but there were no significant changes in the four weeks. However, the expression of vtg was dramatically increased under MT exposure in the 3 and 4 weeks. Compared to AR, vtg is a more sensitive biomarker for long-term(3~4 weeks) MT exposure.This study first provides the full-length cDNA of ERα, ERβ, ERβ1, ERβ2 and AR in Cyprinus carpio var singuonensis, multiple alignments and phylogenetic analysis demonstrated that we have named them correctly. The tissue distribution results showed that ERs and AR MRNAs are broadly expressed in the annlyzed tissues in spite of their greatly varying expression levels in different tissues. ERs and AR mRNA transcription levels following EE2 and MT expose were detected by qRT-PCR, the results demonstrated that four ERs in liver have different responsiveness to EE2 expose, and AR had no significant influence following MT expose, howeve MT may impact to vtg in liver of juveniles.