The CDNA Cloning And Expression Analysis Of Estrogen Receptors In Tanichthys Albonubes

Estrogen receptor (ER) belongs to the nuclear receptor superfamily, and is aligand-dependent transactivation factor. The ER-estrogen complex regulates thetranscription of target genes by interacting with specific estrogen response elements(ERE's), and thus the growth, development and reproduction of organism. The presentstudy was aimed to clone and analyze the expression of ERs in the Tanichthysalbonubes.Based on the previously known 3' end sequence of Tanichthys albonubes ERα(TERα) and T. albonubes ERβ1 (TERβ1), the 5' end sequences of TERαand TERβ1were cloned by the method of 5' RACE. The full-length cDNAs of TERαand TERβ1were 2186 base pairs (bp) and 2609bp, respectively, and their open-reading frame(ORF) encoded putative proteins of 606 amino acid residues. The full-length of T.albonubes ERβ2 (TERβ2) cDNA sequence was also cloned 3'RACE and 5'RACE,which spanned 1939bp, and the ORF encoded a prtative protein of 553 amino acidresidues. The sequence analysis indicated that the three genes were the members ofestrogen receptor family, and their classification, and nomencature were in accordwith other mammals and fish. Like other nuclear receptors, T. albonubes ERs had sixfunctional domains termed A-F from the N-terminus to the C-terminus. The DNAbinding domain (C) ishighly conversed and contains two Zinc fingers, which is acharacteristic of estorgen receptor. The E domain is highly conversed, which is ligandbinding domain.Northern blot analysis showed that the TERαwas mainly expressed in liver offmale T. albonubes, with two transcripts of 8.81kb and 3.63kb, respectively. Bysemi-quantitative reverse transcription-polymerase chains reaction (RT-PCR), theexpression of TERα, TERβ1, and TERβ2 in different tissues in female and male T.albonubes was fruther examined. The mRNA expression of ERs in T. albonubes wastissue-specific and of sexual dimorphism. The expression of TERαwas detectedpredominately in the liver and ovary of female, and in the brain of male.The expression of TERβ1 was detected in all tissues examined, with higher expression levels in the female than in the male. In the female, the highest expressionwas detected in the kidney, which was followed by the liver, intestine, ovary and gillby decreasing order. In the male, the tissue pattern of TERβ1 expression was verysignificant, with very high expression levels in the testis, kidney, intestine and liver,high expression levels in the bone, low expression levels in the brain, eye and muscle,and a very low expression level in the gill.The expression of TERβ2 wasn't detected in the gill, muscle and bone of thefemale, and in the eye, gill and muscle of the male. In the female, the highestexpression level of TERβ2 was detected in the kidney, high expression levels in theliver and intestine, and low expression levels in the ovary, eye and brain. In the male,TERβ2 expression was detected predominantly in the testis, intestine, liver and kidney,with highest expression in the testis, and low expression in the brain and bone.Semi-quantitative RT-PCR analysis showed that the relative expression ofTERβ2 in the liver of male T. albonubes in ethanol control was higher than that in theblank control; the relative expression of TERβ2 in the liver of male T. albonubestreated with 17β-estrodial concentration (100ng/L) was lower than that of ethanolcontrol, and the blank control. These results suggested that the expression of TERβ2could be up-regulated by ethanol, and down-regulated by 17β-estrodial.