Molecular Characteristics And Expression Analysis Of Feminization-1a Gene Related To Sex-determination From Freshwater Pearl Mussel,Hyriopsis Schlegelii

Hyriopsis schlegelii originated from Japan. As a new species of high-quality freshwater pearl mussels, it had important economic value. The study on the sex determination and differentiation could provide theoretical references for the conservation of germplasm resources conservation, population structure, individual development, and the reproduction of Hyriopsis schlegelii. The studies about sex determination genes in vertebrates were reported widely. While, limited information about this field is available in invertebrates especially in freshwater bivalves. In this study, according to the partial fragment of a masculinized feminization-1a gene(Hsfem-1a) from the spermary transcription library, the full-length cDNA of Hsfem-1a were cloned by the RACE PCR technology. Meanwhile, the expression of Hsfem-1a was detected by Real-time PCR. This study could provide the foundation for the further researches on the sex determination and differentiation of Hyriopsis schlegelii.The full length of Hsfem-1a gene was 1356 bp, including 122 bp 5'-UTR, 355 bp 3'-UTR, an open reading frame(ORF) of 879 bp, encoding 292 amino acids. The predicted molecular mass of Hsfem-1a protein was 32.13 kDa; and the theoretical isoelectric point was 7.62; Leucine(Leu) was the highest in the content of amino acids, about 15.1%; it's instablity coefficient was 34.71, the Hsfem-1a protein was a stable protein; did not have signal peptide structure; the deduced protein was hydrophobic. The secondary structure of Hsfem-1a protein mainly contained 39.73%?-helix and 34.25% random coil forming 4 ankyrin repeat(ANK) motifs. The 4 ANK motifs were also found in the tertiary structure of Hsfem-1a protein, which was similar to the prediction result by SMART software. Multiple sequence homology alignment and phylogenetic tree showed that the fem-1a proteins from Hyriopsis schlegelii and Crassostrea gigas shared the highest amino acid identity.The expression of the Hsfem-1a gene were broadly distributed in the11 tissues(female gonad, hepatopancreas, kidney, gills, heart, female gonad, adductoc muscle, intestine, mantle, foot, hemocytes) of Hyriopsis schlegelii. The expression level of Hsfem-1a gene was the highest in the male gonad, followed in the femalegonad, hepatopancreas, intestine, foot, gills, hemocytes, mantle, heart and adductoc muscle. The expression level was the lowest in the kidney. These results suggested that Hsfem-1a may participate in many kinds of physiological functions in different tissues of Hyriopsis schlegelii. The expression of Hsfem-1a in kidney was the lowest expression respectively and so it was taken as a reference, the expression of Hsfem-1a gene in the male gonad was 108-fold. According to these results, the Hsfem-1a mRNA expression in spermary during different development stages in Hyriopsis schlegelii were analyzed by RT-PCR technology. With the development of spermary, the expression of Hsfem-1a increased gradually in the proliferative stage,growing stage, peaking in the mature stage, the expression in the mature stage was7.72 times higher than that in the proliferative stage, and the level in the resting stage decreased. As the maturation of spermary and spermatogenesis, the expression of Hsfem-1a increased gradually, These results suggested that Hsfem-1a played an important role in the development of spermary or the process of spermatogenesis.