Dong-Hee Jo1, Dae-Won Park2, Cheul Min An3, Bo-Hye Nam3, Ji-Min Jeong1, Ju-Won Kim1 and Chan-il Park1*
1Department of Marine Biology & Aquaculture, College of Marine Science, Gyeongsang National University, 455, Tongyeong, 650-160, Republic of Korea
2Gyeongsangnam-do Fisheries Resources Research Institute, Tongyeong, Gyeongnam, 650-947, Republic of Korea
3Biotechnology Research Division, National Fisheries Research and Development Institute, 216 Gijanghaean-ro, Gijang-eup, Gijang-gun, Busan, 619-705, Republic of Korea
Received: 02 July, 2015 Accepted: 04 August, 2015 Published: 05 August, 2015
Chan-il Park , Department of Marine Biology & Aquaculture, College of Marine Science, Gyeongsang National University, 455, Tongyeong, 650-160, Republic of Korea, Tel: +82-55-772-9153; Fax: +82-55-772-9159; E-mail:
Jo DH, Park DW, An CM, Nam BH, Jeong JM, et al. (2015) Analysis on the Molecular Biologic Characteristics and Expression. Int J Aquac Fishery Sci 1(2): 030-034. DOI: 10.17352/2455-8400.000006
© 2015 Jo DH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
C-type lysozymes; Oplegnathus fasciatus; Korea; Molecular biological characteristics
Background and Aim: lysozyme has been known as a significant component of the innate immune system of fish. It is reported that the g-type and c-type lysozymes have been identified in fish. Oplegnathus fasciatus is one of the economically important cultured species, generating higher market value and demands in Korea. Contrary to the heavy consumption of O. fasciatus, however, there has been very limited information on their immune system and immunity genes responding against diseases.
Methods: This study clarified the molecular biological characteristics of cDNA of the C-type lysozyme from O. fasicatus and conducted phylogency analysis. Furthermore, tissue-specific expression analysis of genes verified the roles of c-type lysozyme in general circumstances.
Results: The full-length RbLysC cDNA was 985 bp long and contained an ORF of 432 bp that encoded 143 aa residues, the 5'-UTR of 120 bp and the 3'-UTR of 444 bp. Signal peptide was not found in the terminal and flanking active aspartate region are conserved in RbLysC, and a polyadenylation signal and poly A-tail are present in the 3'-UTR of RbLysC. RbLysC presented the closest distant relationship with sequences from Yellow perch lysozyme C. The highest RbLysC gene expression was observed in the liver, which was about 50-fold relative to that of the PBLs.
The immune system protects organisms from external hazards in tandem with physical barriers like skin and is promptly activated upon the invasion by pathogens. The innate immune system is of more importance to the lower vertebrates like fish than to the higher vertebrate. As one of the most representative parts of innate immune system, lysozyme facilitates the hydrolysis of N-acetyl glucosamine and N-actyl muramic acid in the peptidoglycan layer of bacterial cell walls and destroys the cell walls, thus arresting the spread of bacteria. In gram-positive bacteria, lysozyme is immediately activated, destroying the top layer of peptidoglycan. In gram-negative bacteria, it creates a synergy with positive antibacterial peptide and decomposes external cell walls, thus exposing the peptidoglycan layer.
In addition, lysozyme has been known as a significant component of the innate immune system of fish. It is reported that the g-type and c-type lysozymes have been identified in fish. A c-type lysozyme gene of fish was identified in Rainbow Trout (Oncorhynchus mykiss) first. Since then, a c-type lysozyme gene was found in 13 kinds of fish: Danio rerio, Oryzias latipes, Cyprinus Carpio, Oreochromis aureus, Perca flavescens, Takigfugu rubripes, Scophthalmus rhombus, Scophthalmus maximus, Solea senegalensis, Paralichthys olivaceus, Salmo salar, and Oncorhyncus mykiss and Esox lucius. It was registered in Genbank database [11. Hikima J, Hirono I, Aoki T (1997) Characterization and expression of c-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceus). Mol Mar Biol Biotechnol 6: 339-344.]. A c-type lysozyme gene serves a variety of functions. It not only stimulates digestion, anti-bacterial effects, reproduction, and growth, but also influences tumors [22. Irwin DM, Gong Z (2003) Molecular evolution of vertebrate goose-type lysozyme genes. J Mol Evol 56: 234-242.-55. Liu M, Zhang S, Liu Z, Li H, Xu A, (2006) Characterization, organization and expression of Amphilys C, an acidic C-type lysozyme gene in amphioxus, Branchiostoma belcheri tsingtauense. Gene 367: 110-117.].
A c-type lysozyme gene has easily been expressed in abalones and the invertebrates such as mammals, birds, and reptiles, as well as fish. The gene has been found in other biota, some of which have shown a distinct form or tissue-specific gene expression patterns [66. Rosa M Jimenez-Cantizano, Carlos Infante, Beatriz Martin-Antonio, Marian Ponce, Ismael Hachero, et al. (2008) Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill Scophthalmus rhombus Fish & Shellfish Immunology 25: 57-65.]. In cows, it has been reported that a c-type lysozyme gene is most abundant in intestines and also expressed in a stomach, kidney, respiratory tract, and mammary gland. In mice, it has been reported that a c-type lysozyme is predominantly expressed in small intestines and less predominantly in several tissues [77. Cross M, Mangelsdorf I, Wedel A, Renkawitz R (1988) Mouse lysozyme M gene: isolation, characterization, and expression studies, Proc Natl Acad Sci U S A 85: 6232-6236.].
A g-type lysozyme gene as well as a c-type lysozyme gene has been found in diverse fish species. It is noteworthy that the both types of lysozyme are simultaneously found in Cyprio carpio, Danio rerio, Paralichthys olivaceus, and Scophthalmus rhombus [88. Jiménez-Cantizano RM, Infante C, Martin-Antonio B, Ponce M, Hachero I, (2008) Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill (Scophthalmus rhombus), Fish Shellfish Immunol 25: 57-65.,99. Hikima J, Minagawa S, Hirono I, Aoki T (2001) Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein. Biochim Biophys Acta 1520: 35-44.]. A c-type lysozyme has recently been identified in an invertebrate, Haliotis (Nordotis) discus hannai [1010. Minagawa S, Hikima J, Hirono I, Aoki T, Mori H (2001) Expression of Japanese flounder c-type lysozyme cDNA in insect cells. Dev Comp Immunol 25: 439-445.]. There have been consistent research efforts on the expression patterns and genetic regulation of the two lysozyme genes of some fish. In particular, it was reported that the number of the genes significantly increases after experiment testing bacterial attacks. For Paralichthys olivaceus, the gene was identified most abundantly in head kidney, spleen, and ovary [11. Hikima J, Hirono I, Aoki T (1997) Characterization and expression of c-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceus). Mol Mar Biol Biotechnol 6: 339-344.].
Oplegnathus fasciatus belongs to class of Chordata, Actinopterygii, and perciformes, and is temperate zone fish usually inhabiting in rock zones of coasts. They are distributed in all coastal areas of Korea, the Japanese coastal areas, and the Chinese coastal areas. A male Oplegnathus fasciatus matures faster than a female Oplegnathus fasciatus . Oplegnathus fasciatus is one of the economically important cultured species, generating higher market value and demands in Korea. Contrary to the heavy consumption of Oplegnathus fasciatus, however, there has been very limited information on their immune system and immunity genes responding against diseases.
This study clarified the molecular biological characteristics of cDNA of the C-type lysozyme from Oplegnathus fasicatus and conducted phylogency analysis. Furthermore, tissue-specific expression analysis of genes verified the roles of c-type lysozyme in general circumstances.
Materials and Methods
Molecular characterization of RbLysC cDNA
Been transferred to the laboratory in the purchase rock bream fish farms located in Tongyeong, Gyeongsangnam-do and acclimation were used in the study was 24 hours, feed was not available. Full-length RbLysC (Rock bream Lysozyme C) cDNA was obtained from expressed sequence tags (ESTs) analysis of liver from rock bream stimulated with lipopolysaccharide (LPS) (GenBank accession number AB597292).
Nucleotide sequences of RbLysC were compared lysozyme sequences from other species registered in peptide sequence databases of National Center for Biotechnology Information (NCBI), amino acid homology was found with the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of NCBI. Multiple sequence alignments were analysed using GENETYX ver. 7.0 (SDC Software Development, Japan), positions of the signal peptide were identified using the Signal P program (http://www.cbs.dtu.dk/services/SignalP), phylogeny was inferred using the Mega 4 software and distance analysis using the neighbour-joining method, bootstrap sampling was performed with 2,000 replicates.
Analysis of RbLysC gene expression in various tissues of healthy fish
To evaluate RbLysC gene expression, various tissues including the head kidney, trunk kidney, spleen, liver, intestine, gill and muscle were isolated from three healthy rock breams (Weight 68.5 ± 10 g; body length 14.3 ± 1 cm). Peripheral blood leukocytes (PBLs) were isolated using Percoll density gradients (Sigma-Aldrich, St. Louis, MO, USA), as described previously. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and first strand cDNA synthesis was carried out using a first-strand cDNA synthesis kit (Takara, Shiga, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was performed with SYBR Green Master Mix (Takara, Shiga, Japan) according to the manufacturer's protocol. Quantitative real-time PCR was carried out using cDNA templates for each tissue and specific primer sets for the RbLysC gene (Table 1). Amplification was performed by initial denaturation at 50℃ for 4 min and 95℃ for 10 min, followed by 45 cycles at 95℃ for 30 sec, 60℃ for 30 sec and 72℃ for 30 sec, with a final dissociation at 95℃ for 15 sec, 60℃ for 30 sec and 95℃ for 15 sec. The relative expression of the RbLysC gene was determined by the 2–ΔΔCT method (Livak and Schmittgen, 2001) using β-actin expression as a reference. Data were reported as the RbLysC mRNA levels relative to that of β-actin mRNA, expressed as mean ± standard deviation (SD).
Molecular characterization of RbLysC
The full-length RbLysC cDNA was 985 bp long and contained an open reading frame (ORF) of 432 bp that encoded 143 amino acid (aa) residues, the 5'-untranslated region (UTR) of 120 bp and the 3'-UTR of 444 bp. Signal peptide was not found in the terminal and flanking active aspartate region (64-73aa) are conserved in RbLysC, and a polyadenylation signal (AATAAA) and poly A-tail are present in the 3'-UTR of RbLysC (Figure 1).