Molecular Characterization And Expression Analysis Of Kiss2 And Its Receptor, Gpr54-2 Gene In Japanese Flounder(Paralichthys Olivaceus)

The Japanese flounder(Paralichthys olivaceus) is an important marine economic flatfish in aquaculture. Female Japanese flounders grow faster than the male ones during the growth and development. Thus, the research on sex differentiation and reproductive regulationof Japanese flounder becomes a focused issue. Identi?cation and characterization of the kisspeptin/Gpr54 system is a great breakthrough in Neuroendocrinology which has revolutionized the area of vertebrate reproduction. However, the study of kisspeptin/Gpr54 system in Japanese flounder has not been reported. In this study, we cloned and characterized c DNA of kiss2 and gpr54-2 from Japanese flounder, Paralichthys olivaceus. We also established the expression patterns of kiss2 and gpr54-2 during embryonic and laval development and in adult tissues of Japanese flounder using methods of Real-time PCR, Western-blot and immunohistochemistry. The results are given as follows:1 Molecular characterization of kiss2 and gpr54-2 in Japanese flounderThe length c DNA of kiss2 is 449 bp long, which comprises an 83 bp 3’- untranslated region(UTR) and an open reading frame(ORF) of 366 bp and it encodes a polypeptide of 121 amino acids(aa). It has a predicted signal peptide of 19 aa. The deduced amino acid(aa) seqeunce of Kisspeptin-10 was “FNYNPLTLRF”. The proteolytic cleavage site(GKR) was also found following Kisspeptin-10 region. The full length c DNA of gpr54-2 is 2556 bp and comprises an 899 bp 5’-untranslated region(UTR), an open reading frame of 1143 bp which encodes a polypeptide of 380 aa and a 503 bp 3’- untranslated region and. There are seven transmembrane domains in c DNA of gpr54-2. The phylogenetic analysis showed that Kisspeptin2 of Japanese flounder was located within the clade of Kisspeptin-2 in vertebrates. Gpr54-2 of Japanese flounder was located within the clade of Gpr54-2 in fishes. The deduced amino acid sequence of Kisspeptin2 shared 76%, 74%, 69%and 28% similarity with Morone saxatilis, Oreochromis niloticus, Solea senegalensis and Danio rerio. The deduced amino acid sequence of Gpr54-2 shared 98%, 93% and 88% similarity with Hippoglossus hippoglossus, Solea senegalensis and Danio rerio.2 Expression patterns of kiss2 and gpr54-2 in adult tissues of Japanese flounder Real-time PCR results unveiled the fact that the highest level of kiss2 m RNA was detected in brain, followed by liver, gill, and gonads. A relatively low kiss2 m RNA was observed in heart, muscle and testis, while kiss2 transcripts were not detected in kidney and stomach. The gpr54-2 m RNA was observed in all detected tissues. Its expression level, however, varied much among different detected tissues. The highest expression of gpr54-2 m RNA was observed in brain, followed by gonads. Its expression level in ovary was a little higher than that in testis. The expression level of gpr54-2 m RNA in other tissues was relative low. The high expression of kiss2 and gpr54-2 in brain suggested they may be involved in nervous system and reproduction development of Japanese flounder3 Expression patterns of kiss2 and gpr54-2 during embryonic and laval development of Japanese flounder The kiss2 transcripts were in a low level at embryonic stages, especially, not detected at unfertilized stage. After hatching, the expression of kiss2 m RNA increased and reached its peak at 29 dph. The gpr54-2 m RNA was detected in all embryonic and larval development stages. Its expression level was relative low during early embryonic stages. The expression level of gpr54-2 m RNA increased during larval development stages and reached its peak at 36 dph. 4 Expression of Gpr54-2 protein in in adult tissues of Japanese flounderWestern-blot result revealed that Gpr54-2 protein was mainly observed in brain,testis and ovary and it was weakly expressed in liver. Immunohistochemical results showed weak signal of Gpr54-2 protein in examined tissues.This study laid foundation for further research of kiss2 and gpr54-2 in sexual differentiation and reproduction of Japanese flounder.