Nibea coibor growth hormone gene: its phylogenetic significance, microsatellite variation and expression analysis.

ABSTRACT

The growth hormone (GH) gene has been characterized for a number of fishes and used to establish phylogenetic relationships and as a candidate gene for studies of genetic variation in connection with growth traits. In this study, we report the genomic structure of Nibea coibor GH (designated as ncGH) including its 5'-flanking region, being cloned by homology-cloning and chromosome walking methods. The ncGH gene spans approximately 3.0 kb and consists of six exons and five introns, as found for all cloned teleost GH genes with the exception of carps and catfish. The 5'-flanking region contains consensus sequences for a TATA box, a CRE, a pit-1alpha, a TRE, two HNF-3, a ERE and a GRE. Five microsatellites are identified in the ncGH gene and three of them are polymorphic marker. The open reading frame (ORF) of ncGH is 615 bp in length encoding a polypeptide of 204 amino acids with an estimated molecular mass of 23.04 kDa and theoretical isoelectric point of 7.79. The precursor of ncGH consists of a 17 amino-acid signal peptide and a 187 amino-acid mature peptide. The four Cys residues are located at conserved positions (Cys(69), Cys(177), Cys(194) and Cys(202)), and One possible site for N-glycosylation (Asn-X-Ser/Thr motif) is present at Asn(201). The coding region sequence of ncGH is used to align with the sequences of 18 other species from Percoidei and one species from Anabantoidei using Clustal X. A matrix of 612 bp was used to construct the phylogenetic trees using neighbor-joining and maximum parsimony methods. The phylogenetic trees by two methods are identical in most of the clades with high bootstrap support. Every family all forms independent monophyly on the phylogenetic trees, in the family, the different species also forms the monophyly according to the different genera. The results are also identical to those from morphological data, and demonstrated that the GH gene is very suitable for phylogenetic relationship analysis of Percoidei. To validate the predicted exon/intron boundaries, ncGH cDNA is cloned using RT-PCR, and tissue distributions are investigated using semi-quantitative RT-PCR method. The results indicate that the predicted exon/intron is correct, the ncGH mRNA are mainly expressed in pituitary, and weakly expressed in ovary, brain, liver, gill, intestine, muscle and hear, but not expressed in spleen and kidney.