Study On Molecular Characteristics And Sex-regulation Function Of Sox9 In Freshwater Peal Mussel,Hyriopsis Schlegelii

Sox9 gene plays a basilic role in gender determination and differentiation,early embryonic development,nervous system development,cartilage and the formation of multiple organs.The molecule mechanism of Sox9 may be different from vertebrates in the gonadal development and and gametogenesis of shellfish.The full-length cDNA of Sox9 in Hyriopsis schlegelii(Hs-Sox9)was cloned using RACE-PCR technology.The Hs-Sox9 protein and Hs-Dmrt1 protein were expressed in prokaryotic cells and purified by Ni-exchange column.The Hs-Sox9 promoter sequence was cloned using the Genome-Walker technology.The expression of Hs-Sox9 genewas analyzed in different tissues of Hyriopsis schlegelii.The affinity of Hs-Sox9 protein and Hs-Dmrt1 gene promoter was analyzed.Hyriopsis schlegelii were challenge to environmental factors(temperature and hormone)and the expression of Hs-Sox9 and Hs-Dmrt1 genes were analyzed.The full-length cDNA of Hs-Sox9 gene is 1654 bp,whose open reading frame(ORF)sequence is 1398 bp and encodes 466 amino acids.It has a highly conserved HMG(included 70 amino acids)and Sox-N domain.The Sox9 amino acid sequences from 14 different species were compared and a phylogenetic tree was constructed.The results showed that Hyriopsis schlegelii had the closest relationship with Chlamys farreri and Pinctada margaritifera.The promoter sequences of Hs-Sox9 and Hs-Dmrt1 genes were cloned and the lengths was 1156 bp and 1363 bp.In addition to the basic features shared with common promoters,the promoters of these two genes contain sexrelated gene binding sites such as SRY and Sox9.The Hs-Sox9 protein was analyzed using bioinformatics.Serine is the most abundant amino of the protein,whose proportion was 12.9%.The molecular weight is 52308.51.The p I is 6.71.The protein,with 22.8% alpha helix,4.3% extended chain,and 72.9% random coils,which average hydrophilicity coefficient is-0.989,belongs to the intracellular hydrophilic protein.After prokaryotic expression,the Hs-Sox9 and HsDmrt1 proteins were purified by Ni-exchange column.It was found that the Hs-Dmrt1 protein had no affinity with Hs-Sox9 and Hs-Dmrt1 promoters by gel retardation analysis.The Hs-Sox9 promoter had certain affinity with both proteins.These results showed that the expression of Hs-Dmrt1 gene might be regulated by the Hs-Sox9 protein in the sex determination and differentiation.The expression of Hs-Sox9 was significantly high in the testis The Hs-Sox9 gene was highly expressed in the testis,mantle and gill in male.It was highly expressed in the mantle and gill in female.The expression of Hs-Sox9 and Hs-Dmrt1 gene was restrained by the increase of temperature.The expression patterns of the Hs-Sox9 and the Hs-Dmrt1 gene challenged to environment factors indicated that these two genes may participate in the common signaling pathway.And the results are in line with the conclusion of gel retardation.