Functional Divergence of the N-Lobe and C-Lobe of Transferrin Gene in Pungitius sinensis (Amur Stickleback).

Transferrin is an important iron-binding glycosylated protein and plays key roles in iron-binding and immune response. Here, a 2037-bp open reading frame was obtained from our previous transcriptome sequencing data of Amur stickleback, which encoded a 679 amino acid putative transferrin protein harbored obvious N-lobe and C-lobe domains. The tissue-specific expression pattern showed that the transcript was detected in a variety of tissues, with the highest signal in liver. Moreover, Streptococcus iniae pathogen stimulation can increase the expression level of this transcript, implying important immune properties for organisms. Next, N-lobes and C-lobes were obtained from 45 fish species. The phylogenetic tree showed that N-lobes and C-lobes were in two different evolutionary branches, and they had different motif composition. Functional divergence indicated a higher evolutionary rate or site-specific alteration among the N-lobe and C-lobe groups. Ka / Ks value of C-lobe group was relatively higher than that of N-lobe group, indicating a faster change rate of C-lobe sequences in evolution. Moreover, some sites experiencing positive selection were also found, which may be involved in the iron- or anion-binding, pathogen resistance and diversification of transferrin protein. Differential iron-binding activity was also detected between N-lobe and C-lobe of Amur stickleback transferrin protein with Chrome Azurol S assay. Compared with the C-lobe, the N-lobe showed stronger growth inhibitory activity of Escherichia coli , implying their potential antibacterial properties. This study will give a reference for subsequent research of transferrin proteins.