Cloning, Expression And Functional Analysis Of Two C-type Lectins Genes In Roughskin Sculpin (Trachidermus Fasciatus)

Roughskin sculpin (Trachidermus fasciatus) is a catadromous fish with seawater-freshwater migratory habit. In the history, Roughskin sculpin distributes widely in the coastal areas of China, but now wild population of Roughskin sculpin has drastically declined because of environmental pollution. Consequently, Roughskin sculpin was listed as critically endangered in Category II of the National Key Protected Wildlife List in 1988. Aquaculture has become an important strategy for recovering its population in the wild. However, stressful conditions facilitate the appearance and diffusion of disease in fish aquaculture. Roughskin sculpin immune response to pathogen exposure should be studied thoroughly in order to deal with the disease. C-type lectins are a protein family which can bind carbohydrates in a calcium-dependent manner. They Play roles as the receptors to recognize the pathogen pattens in the immune system, and have other function as well.. We colend two transmembrane C-type lectins to better understanding the function of C-type lectins in Roughskin sculpin.TfCD209 was cloned from Roughskin sculpin. The cDNA of TfCD209 was 1042 bp, with a 5’UTR of 256 bp, a 3’UTR of 15 bp, and an open reading frame (ORF) of 771 bp that encoded a protein of 256 amino acids with the predicted molecular weight of 29078.7 and isoelectric of 5.45. It had a transmembrane and a carbohydrate-binding domains, with a carbohydrate-binding site (EPN motif), a conserved motif WIGL, and four conserved disulfide-bonded cysteine residues. It had two a-helixes and five P-sheets; Neighbor-joining tree showed that TfCD209 clustered to CD209 from the other fishes in accordance to their traditional taxonomy. qRT-PCR analysis revealed that TfCD209 was constitutively expressed in all detected tissues, with a relatively rich amount of mRNA in the liver. Its expression was up-regulated in the four detected tissues (liver, gill, skin and spleen) challenged with Vibrio anguillarum. Recombinant TfCD209 CTLD protein bound to some Gram-positive and Gram-negative bacteria in a calcium-independent manner. These results suggested that TfCD209 might be involved in the innate response as a PRR.TfCD302 was cloned from Roughskin sculpin. The cDNA of TfCD302 was 2195 bp, with a 5’UTR of 62 bp, a 3’UTR of 1448 bp, and an open reading frame (ORF) of 735 bp that encoded a protein of 244 amino acids with the predicted molecular weight of 27165.8, and isoelectric of 4.69. It contained a signal peptide, a transmembrane and a carbohydrate-binding domains, without carbohydrate-binding site. It still had the four conserved disulfide-bonded cysteine residues. It had two α-helixes and five β-sheets; Neighbor-joining tree showed that TfCD302 clustered to CD302 from the other fishes in accordance to their traditional taxonomy. qRT-PCR analysis revealed that TfCD302 was constitutively expressed in all detected tissues, with a relatively rich amount of mRNA in the spleen. Its expression was up-regulated in the four detected tissues (liver, gill, skin and spleen) challenged with Vibrio anguillarum. Recombinant TfCD302 CTLD protein bound to some Gram-positive and Gram-negative bacteria in a calcium-independent manner. These results suggested that TfCD302 might be involved in the innate response as a PRR.