Genomic and expression analysis of a solute carrier protein (CcSLC25a5) gene from Cyprinus carpio Linnaeus

Abstract

Using the Genefishing method, we identified seven potential regulatory genes involved in the process of scale morphogenesis in fishes. We further characterized a novel solute carrier protein gene (CcSLC), from the common carp which is differentially expressed in mirror carp and Jianli. The ORF encodes a peptide of 298 amino acids with a molecular mass of 31.5 kDa and a theoretical isoelectric point of 7.49. ScanProsite analysis indicated that it is a putative solute carrier protein that contains a substrate binding site. CcSLC was detected in carp embryos by in situ hybridization in the 70%-epiboly, 6-somite, and 14-somite embryonic stages. Gene expression stopped at the long pec stage. However, CcSLC25a5 was re-expressed during the initiation of scale formation in the regions that were scale covered. These findings provide novel insights into the features of early carp embryo and scale development.

Keywords: Cyprinus carpio Linnaeus; Embryo development; Scale development; Scale initiation; Solute carrier protein.

Figure 1

Differential gene expression detected in the Genefishing assay using primers ACP28 and ACP29. The PCR products from the genefishing assay were separated by agarose gel electrophoresis. Gene products that are differentially expressed in the mirror carp (KL-2) and common carp (JL-2) are shown by green arrows. DL5000 indicate molecular markers.

Figure 2

Comparing of the amino acid sequences deduced from six carp ANT2 transcripts. Dotted lines under the sequences indicate the positions of six putative transmembrane helices (Klingenberg 1989) labeled segments I-VI. Three repeats of the mitochondrial carrier protein signature (Bof et al. 1999) are shown by dotted boxes. A substrate binding site is highlighted with shaded boxes. Amino acids that differ between the transcripts are indicated in blue.

Figure 3

The gene structure of the carp, zebrafish, and human ANT genes. Coding regions in the exons are represented by black boxes. Introns are represented by solid lines.

Figure 4

A phylogenetic tree of carp and mammalian ANT isoforms constructed using the neighbor-joining methodology. The evolutionary distance between amino acid substitutions was estimated using Kimura’s two parameter-method (Kimura 1980). The scale along the bottom axis shows the evolutionary distance of amino acid substitutions per site.

Figure 5

The CcSLC25a5 expression pattern by whole-mount in situ . Panels A-G show the expression pattern of CcSLC25a5 genes in different developmental stages. (A) 4.5 haf (1/3 epiboly), (B) 6 haf (shield), (C) 10 haf, (D) 12 haf, (E) 16 haf, (F) 24 haf, and (G) 48 haf after fertilization, respectively. (H) SLC25a5 is expressed specifically in the regions where the scales were developing in the skin. (I) There is no hybridization signal detected using the sense probe as the negative control.