Miiuy croaker transferrin gene and evidence for positive selection events reveal different evolutionary patterns

Abstract

Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved.

Figure 1. A comparison of the miiuy croaker TF gene structure with the previously published gene structures of other fish species and mammals.

Exons were represented by boxes. The length of the exons was indicated on the top of boxes and the length of introns was indicated under the line below each gene structure. And the same color of each column represents that the length of exon was the same.

Figure 2. Expression analysis of MIMI-TF by relative quantitative real-time PCR in liver (A), spleen(B) and kidney (C) during 6, 12, 24, 36, 48, and 72 h of induction with V. anguillarum, and (D) Expression of TF gene in various tissues (heart, muscle, kidney, eye, gill, intestine, brain, spleen, fin and liver of uninfected miiuy croaker.

Deviation bars represented the standard errors (± the SD/SE) of three experiments at each time point.

Figure 3. Phylogenetic tree of the nucleotide sequences of transferrin proteins from 38 taxa.

The phylogeny of the sequences was estimated using the Bayesian inference implemented in the software MrBayes.

Figure 4. Three-dimensional structure of human transferrin proteins.

Images depicted represent four different perspectives (the angles of view are 0°, 90°, 180°, and 270°for the front, back and side via rotation in 90°increments around the vertical axis) of the surface of a space-filled model of human structure.