Characterization of tilapia FSHβ gene and analysis of its 5′ flanking region

Abstract

The objective of the current study was to unveil molecular mechanisms underlying transcriptional regulation of the FSHβ gene expression in the pituitary of tilapia (Oreochromis mossambicus). The full-length sequence of tilapia FSHβ (tFSHβ) gene was determined. Its transcriptional unit (2.7 kb) exhibits the conserved genomic organization, i.e. three exons and two introns. Primer extension and RT-PCR analysis revealed heterogeneity of the tFSHβ transcripts, due to alternate mRNA splicing and multiple initiation sites for transcription. Examination of the 5′ flanking region (5′FR) of the tFSHβ gene identified potential CAAT and TATA promoter proximal elements as well as several sequences of cis-acting motifs known to dictate inducible and tissue-specific transcriptional regulation in other gonadotropin genes. Chimeric constructs containing 1.7 kb of the tFSHβ 5′FR fused to a luciferase (LUC) reporter gene were transiently transfected into primary culture of tilapia pituitary cells. The tFSHβ-LUC construct was efficiently expressed under basal conditions and was rapidly induced by GnRH stimulation. Our data indicate that the 5′FR contains a functional promoter, which is responsive to GnRH treatment. In addition, 5′ deletion analysis showed that the 1.7 kb, DNA sequence of the FSHβ 5′FR encompasses both positive and negative regulatory elements.