Molecular characterization and expression analysis of olive flounder (Paralichthys olivaceus) phospholipase C gamma 1 and gamma 2.

ABSTRACT

Phospholipase C gamma 1 and gamma 2 (PLCG1 and PLCG2) are influential in modulating Ca2+ and diacylglycerol, second messengers involved in tyrosine kinase-dependent signaling, including growth factor activation. Here, we used RACE (rapid amplification of cDNA ends) to clone cDNA encoding PLCG1 (PoPLCG1) and PLCG2 (PoPLCG2) in the olive flounder (Paralichthys olivaceus). The respective 1313 and 1249 amino acid sequences share high identity with human PLCG1 and PLCG2, and contain the following domains pleckstrin homology (PH), EF-hand, catalytic X and Y, Src homology 2 (SH2), Src homology 3 (SH3), and C2. Phylogenic analysis and sequence comparison of PoPLCG1 and PoPLCG2 with other PLC isozymes showed a close relationship between the two PLCGs, supported by structural analysis. In addition, tissue expression analysis showed that PoPLCG1 was expressed predominantly in the brain, eye, and heart, whereas PoPLCG2 was expressed principally in gills, esophagus, spleen, and kidney. Following stimulation with LPS and Poly IC, PoPLCG expression was compared with the expression of inflammatory cytokines IL-1ß, IL-6, and TNF-α via reverse transcription-PCR and real-time quantitative PCR. Our results suggest that PoPLCG isozymes perform a critical immune function in olive flounder, being active in pathogen resistance and the inflammation process.