Sequence analysis and expression regulation of rbp4 by 9-cis-RA in Megalobrama amblycephala.

ABSTRACT

Retinol-binding protein 4 (rbp4) is mainly synthesized in the liver, where it binds retinol and then enters the bloodstream, delivering retinol to cells. The full-length cDNA coding rbp4 was cloned from Megalobrama amblycephala. The amino acid sequence showed strong homology with the homologues of other vertebrates, and all structural and functional domains were highly conserved. The mRNA levels in different tissues and development stages detected by quantitative real-time PCR revealed that M. amblycephala rbp4 was highly expressed in liver (P < 0.001), but the lower levels were also detected in eyes, kidney, intestine, and spleen. During the different development stages, the rbp4 mRNA appeared until 28 hours post-fertilization (hpf), underwent a slight drop, and then gradually increased after 50 hpf. In addition, the promoter sequence of M. amblycephala rbp4 was obtained using thermal asymmetric interlaced PCR. Two single nucleotide polymorphism sites (-385A>G and -329C>T) were found in the promoter. Transfection with recombinant plasmids of two different haplotypes (GT, AC) showed that 9-cis-retinoic acid (RA) increased the promoter activity, but the AC haplotype was more sensitive to RA.