牙鲆两种维生素D受体基因克隆、组织表达与结构比较研究

he action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, is mediated by their specific receptor, VDR, which controls the expression of hormonesensitive genes. VDR is an important factor in calcium homeostasis and bone development. To reveal the function of vitamin D and VDR of fish as well as the molecular structure of VDR, it is important to compare different subtype VDR molecules from the flounder. In contrast to the tetrapods, in which only a single type of VDR gene has been identified, Japanese flounder was found to have two subtypes of VDR. Flounder VDR a and b share 82% identity at the nucleic acid level and 84% identity at the amino acid level. Expression profile analysis by a reverse transcription-PCR (RT-PCR) technique detected the expression of VDR in 5 tissues. The results shows that Japanese flounder VDRa and VDRb mRNA are both expressed in skin, intestine, gill and brain. In addition, VDRa in skin and VDRb in intestine weakly expressed but both VDRa and VDRb mRNA are undetectable in liver. The 3'-untranslated region following the coding region of VDRa and VDRb were rich in A+T. 26 predicted stem-loop structures in VDRa and 23 in VDRb resided in the transcribed mRNA sequence corresponding to this region. The stem-loops were tandem organized and this region formed a complicated cloverleaf like secondary structure with a free energy of -113.4 kkal/mol in VDRa and -92.5kkal/mol in VDRb. Homology modeling results show that the VDRa and VDRb in this study had similarity three-dimensional structure but differences still exist. VDRa and VDRb were composed of different amounts of alpha regions, turns regions and beta sheet regions. In general, different expression levels in different tissue and differences in three-dimensional structure between VDRa and VDRb indicate that the function or roles were different in VDRa and VDRb.