The C- And G-type Lysozyme Genes And Their Function In The Japanese Eel Anguilla Japonica

Lysozyme is a kind of enzyme which cleaves β-(1, 4)-glycosidic bond between Nacetylglucosamine and N-acetylmuramic acid in peptidoglycan, an important component of bacterial cell walls, and plays an important role in host immunity. Based on the genomic database of the Japanese eel(Anguilla japonica), we amplified 2 types of lysozyme genes, with 1 C-type and 4 G-type, named as AJLys C, AJLys G1, AJLys G2, AJLys G3, AJLys G4, consisting of 809, 732, 1352, 1177 and 731 nucleotides, coding for 143, 193, 185, 185 and 187 amino acids, respectively. AJLys C and AJLys G1 have signal peptides consisting of 15 and 19 amino acids, respectively, and the other three do not have signal peptides. The gene structure of AJLys C, AJLys G2, AJLys G3, AJLys G4 are similar in the exon-intron structure of lysozymes in other species of fish, with 4 exons for the C-type and 5 exons for the G-type. However, the gene structure of AJLys G1 is different from lysozymes in other fishes, with 6 exons, representing the first report of this type in fish. When compared with other species, it was found that the second catalytic residue, Asp, in AJLys G1 is missing. AJLys C shares strong identity with C-type lysozyme of many other species, for instance being 72.7% with that in Japanese flounder(Paralichthys olivaceus). Three of the 4 G-type lysozymes, AJLys G2, AJLys G3, AJLys G4, share much higher identity with each other and with that of other species, while AJLys G1 does not share high identity with neither G-type lysozymes of other species nor the former three, with the identity being lower than 50%.The rt-q PCR results showed that all the five genes were expressed in all the 12 organs/tissues examined. The C-type lysozyme gene was expressed at highest level in stomach, and high in immune related tissues. The expression of other four G-type lysozymes was different in different organs/tissues. For instance, AJLys G1 was highly expressed in skin and muscle, while AJLys G2 in blood, head kidney, body kidney and gill. At 48 hours after inoculated with Edwarsiella tarda, all the five genes were up-regulated, especially in blood, intestine and head kidney. In addition, we also constructed the eukaryotic expression of AJLys G2, AJLys G3 and AJLys G4 in order to analyze their antimicrobial activity. The recombinant proteins expressed in EPC cells showed obvious activity in inhibiting the growth of Micrococcus lysodeikticus, and further study should be extended to examine their role in inhibiting pathogenic bacteria of the Japanese eel.