Partial Characterization of the Genomic Organization of the GnIH Gene in Zebrafish Using Genome Walking

Abstract

The gonadotropin-inhibitory hormone (GnIH) gene is an important component of the reproductive neuroendocrine system in vertebrates, and its genomic characterisation can provide useful information for comparative reproductive biology. The present study aimed to partially elucidate the genomic organisation of the GnIH gene in zebrafish (Danio rerio) using a PCR-based genome-walking approach. Adult zebrafish of the AB strain were maintained under controlled laboratory conditions with a 14-hour light:10-hour dark cycle, a water pH of 7.5, and a temperature of 28.5 °C. Genomic DNA was isolated from fin tissue and used to construct adaptor-ligated genomic DNA libraries after restriction digestion with DraI, EcoRV, PvuII, and StuI. Gene-specific primers designed from the known exon 2 region were used in primary and nested PCR reactions with adaptor-specific primers to amplify unknown upstream genomic regions. The amplified fragments were sequenced bidirectionally, enabling the partial identification of intron 1 of the zebrafish GnIH gene. The resulting sequence information provides a useful basis for further characterisation of exon-intron boundaries and regulatory regions associated with the GnIH locus. This study demonstrates the applicability of PCR-based genome walking for retrieving unknown genomic regions adjacent to known GnIH sequences in zebrafish.