강도다리, Platichthys stellatus lysozyme의 분자학적 특성과 발현 분석

Abstract

Flatfish have arisen world wide as important candidates for diversification in aquaculture. In Korea, the starry flounder, Platichtys stellatus is considered as an alternative species to olive flounder, Paralichthys olivaceus, according to its high commercial value. The aquaculture of starry flounder had begun on east of Korea from 2006, the first mass mortality occurred in 2007 by infection of Streptococcus parauberis, which is gram-positive bacteria with single peptidoglycan layer on the cell wall. Fish innate immune response is activated by granular leukocyte and macrophage at the intial stage of infection. Lysozyme is an important molecules in the innate immune system and other inner organs. The key role is catalysis of β-1, 4-glycosidic bond hydrolysis of peptidoglycan layer. Therefore, lysozyme has been considered as one of the most effective defense factor against bacterial infection, including S. parauberis. Lysozyme activity of skin mucus and serum was investigated after abdominal injection with S. parauberis and physiological saline as control. The lysozyme activity of skin mucus increase immediately after injection, and reached peak level after 8 hour post-injection, but it decrease gradually thereafter. Control group activity did not show any significant change. Lysozyme activity of serum reached the highest level at 4 h post infection. At 5 day of post-injection, the activity level of lysozyme decrease, still kept higher level than control even if decreased. The control group activity increase gradually, after injection. In this study, Chicken (C)- and Goose (G)-type lysozyme cDNAs were cloned and characterized. Also the tissue expression profiles of each type of lysozyme genes were investigated in the normal starry flounder and successively after injection with S. parauberis . For cloning of two type lysozyme genes, partial fragments were amplified using degenerated primers, and Rapid Amplification of cDNA Ends (RACE)-PCR was performed. C-type lysozyme cDNA full- length is 625 bp, including a 5'-untranslated region (UTR) of 28 bp and a 3'-UTR of 165 bp. The 432 open reading frame (ORF) encodes a protein of 143 deduced amino acid, including 8 conserved cysteine residues and 2 catalytic residues, Glutamic acid (Glu) and Aspartic acid (Asp). G-type lysozyme cDNA full- length is 870 bp, including a 5'-UTR of 129 bp and a 3'-UTR of 174 bp. The 567 ORF encodes a protein of 188 deduced amino acid, including 2 catalytic residues Glu and Asp. The semi quantitative reverse transcription (RT)-PCR was used to determine the tissue expression profiles of lysozyme genes. In normal state of starry flounder, C-type lysozyme mRNA was expressed in spleen, liver and gill, and G-type lysozyme was expressed in various tissue including heart, kidney, spleen, gut, intestine, liver and gill. Lysozyme C was up-regulated after 72 h post injection with S. parauberis in kidney and spleen, but not increse in liver of starry flounder. It Lysozyme G was increse after 4 h post injection in kidney, spleen and liver, and decrese after 72 h post injection.