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

Two genes related to sex determination named Hsfem-1b and Hsfem-1crelatively were cloned from freshwater mussle, Hyriopsis schlegelii, using SMARTrapid amplification of cDNA ends and nested PCR technology.The full-length cDNA sequence of Hsfem-1b is2072bp (GenBank accessionnumber：KJ755434), consisting of a5’-terminal untranslated region (UTR) of50bp,an open reading frame of1908bp, a3’-terminal UTR of114bp with a28bp poly (A)tail and a tailing signal (ATTAAA). The Hsfem-1b cDNA encodes a polypeptide of635amino acids without signal peptide. The predicted molecular mass of the HsFem-1b is71.54kDa with a theoretical isoelectric point of6.05. The C-terminal of HsFem-1b protein has a highly conserved amino acid sequence: Pro-X-X-Leu-X-X-Phe-X-X-X-His (X can be a random amino acid). TheHsFem-1b protein contains8ANK(Ankyrin repeat motif) repeats by using SMART prediction. We also predicted itsthree-dimensional structure by using SWISS-MODEL workspace. The results showedclearly8ANK motifs, which was consistent with the SMART prediction.The full-length cDNA sequence of Hsfem-1c is2328bp (GenBank accessionnumber：KJ755433), consisting of a5’-UTR of137bp, an open reading frame of1869bp, and a3’-terminal UTR of322bp with a25bp poly (A) tail and a tailingsignal (AATAAA). The Hsfem-1c cDNA encodes a polypeptide of622amino acidswithout signal peptide. The predicted molecular mass of HsFem-1c is69.95kDa withthe theoretical isoelectric point of7.05. The C-terminal of HsFem-1c protein also hasa highly conserved amino acid sequence: Pro-X-X-Leu-X-X-Phe-X-X-X-His (X canbe a random amino acid). There are9ANK repeats in HsFem-1c proteinpredicted byusing SMART. We also predicted its three-dimensional structure by using SWISS-MODEL workspace. The result showed clearly9ANK motifs, which was consistentwith the SMART prediction. The expression of Hsfem-1b and Hsfem-1c mRNA in Hyriopsis schlegelii wereanalyzed by fluorescent real-time quantitative PCR. The results showed that themRNA expression of Hsfem-1b and Hsfem-1c had obvious tissue specificity. Theexpression level of Hsfem-1b and Hsfem-1c in testis was the highest, which was72.35times and77.87times higher than that of kidney respectively. Followed by the liver,the intestine, the ovary, the mantle, the gills and adductor muscle. The expressionlevel of Hsfem-1b and Hsfem-1c was the lowest in the kidney. We found theexpression levels of Hsfem-1b and Hsfem-1c gene are in different trend from thereaserch about mRNA expression of different ages Hyriopsis schlegelii testis. Withgonadal maturation and spermatogenesis, the expression of Hsfem-1b gene graduallyincreases, indicating it plays an important role in the process of spermatogenesis orgonad development. The expression of Hsfem-1c gene was significantly up-regulatedin2-ages Hyriopsis schlegelii testis, it up to6.77times of one age, However, theexpression of the3-4ages Hyriopsis schlegelii testis dropped back, and in age5,theexpression level has again increased significantly to7.67times of one age. Hyriopsisschlegelii gonadal cells begin to grow into testis or ovary, indicating Hsfem-1c genemaybe involved in the process of Hyriopsis schlegelii male gonadal sexdifferentiation, and associated with spermatogenesis.pET32-HsFem-1b and pET32-HsFem-1c prokaryotic expression systems wereconstructed by double digestion. The correct prokaryotic expression plasmidschecked by sequencing were then transformed into E.coli (DE3). After IPTGinduction, the two recombinant proteins were successfully expressed. The HsFem-1brecombinant protein form insoluble inclusion bodies. The inclusion bodies weredissolved by the denaturant and purified by using the native Ni2+affinitychromatography after refolding using gradient dilution. HsFem-1c recombinantprotein is mainly in the form of soluble proteins, which access to the single targetrecombinant protein directly by nickel column. Rabbit polyclonal antibody was prepared using purified HsFem-1c recombinantprotein. The serum antibody titers measured by ELISA was higher than512000,indicating a high-titer antisera obtained for HsFem-1c. Western blotting showed thatHsFem-1c recombinant protein was able to combine with rabbit polyclonal antibodyspecifically. We also prepared frozen sections of gonad for immunofluorescentanalysis of HsFem-1c using the rabbit polyconal antisera. We found HsFem-1cprotein was mainly expressed in the cytoplasm of the gonad cells.