Characterization and phylogenetic analysisof a g-type lysozyme gene variant from the skin mucus of grass carp (Ctenopharyngodon idella)

The g-type lysozyme is one of the three major diverse lysozyme types recognized in the animal kingdom including fish. Using the RT-PCR technique, a 555 bp cDNA fragment encoding a g-type lysozymewas isolated from the skin mucus of Ctenopharyngodon idella using homolog primes. The cDNA named Ci-Kh, codes for 185 amino acids with a predicted molecular weight of 20.49 kDa and theoretical pI of 9.13. The sequence consists of one cysteine residue with no predicted signal peptide. Domain analysis showed e-value of 3.74e-107 with the conserved domain of lysozyme-like superfamily (cd01021) between amino acid residues 12 to 184. Multiple alignment with the lysozyme genes from other fish species revealed that this protein have a goose egg white lysozyme (GEWL) domain containing two conserved catalytic residues (Glu73 and Asp97) and N-acetyl-D-glucosamine binding site (Glu73, Asp85, Asp97, Tyr100, His101, His102, Ile119, Tyr147, Asn148). Protein structure prediction software revealed a prediction of 58% and 42% of a-helical and random coils for the coding sequence of Ci-Kh, respectively.The 3D model of Ci-Kh revealed that this protein was mainly composed of five main helices and random coils. Phylogenetic analysis indicated that Ci-Kh matched the group of five grass carp g-type lysozyme transcript variants with 86-99% similarity. Among the five variants of this gene, Ci-Kh sequence had the highest and lowest genetic distance with C. idella variant X2 (8.6%) and X3 (1.1%) sequences, respectively. We conclude that Ci-Kh as a different variant of g-type lysozyme cannot be ruled out.