Molecular characterization and expression pattern of two zona pellucida genes in half-smooth tongue sole (Cynoglossus semilaevis)

Abstract

Zona pellucida (ZP) proteins are glycoproteins in fish chorion that are encoded by multiple gene families, mainly including zp1, zp2, and zp3. In the present study, we cloned two zp genes of half-smooth tongue sole, Cynoglossus semilaevis, using reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The two genes were demonstrated to belong to zp3 isoforms by utilizing BLAST, alignment of amino acid sequence, and phylogenetic analysis. The two genes were named C. semilaevis zp3a and C. semilaevis zp3b. C. semilaevis zp3a encoded 519 amino acid protein, including a signal peptide and a ZP domain with 257 amino acids, which was 62% identical to the medaka ZPC5. Incontrast, C. semilaevis zp3b encoded a 313 amino acid protein, including a signal peptide and a ZP domain with 233 amino acids, which was 63% identical to the medaka (Oryzias latipes) ZPC1. Alignment analysis showed that the ZP domain of the two ZP proteins contained eight conserved cysteines. RT-PCR indicated that the C. semilaevis zp3a was highly expressed in the ovary and kidney of females, and weakly expressed in female muscle and spleen. The C. semilaevis zp3b mRNA was expressed in several tissues of females,including a high expression level in the ovary and kidney, and a relatively low expression level in the heart,brain, muscle, spleen, gill, and intestine. Interestingly, the zp3b mRNA was slightly detected in the testis and kidney of males. Therefore, molecular cloning and characterization of zp genes could lay a foundation for our understanding of the regulation of zp gene evolution and the regulatory mechanism of fertilization.