title: Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus.
abstract: The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5'-untranslated region (UTR) of 17 bp, a 3'-UTR of 76 bp, and a stop codon TAA in 3'-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.
authors: Marine Applied Microbes and Aquatic Organism Disease Control Lab, Department of Aquatic Biomedical Sciences, School of Marine Biomedical Sciences & Marine and Environmental Research Institute, Jeju National University, Jeju 690-756, South Korea. rhari123@yahoo.com
authors: Ramasamy
authors: R
authors: Harikrishnan
authors: Ju-Sang
authors: JS
authors: Kim
authors: Man-Chul
authors: MC
authors: Kim
authors: Chellam
authors: C
authors: Balasundaram
authors: Moon-Soo
authors: MS
authors: Heo
mesh_terms: D000818:Animals; D001483:Base Sequence; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D015723:Gene Library; D007113:Immunity, Innate / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D010473:Perciformes / Q000235:genetics*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D036341:Intercellular Signaling Peptides and Proteins