Cloning, Sequence Analysis And Tissue Expression Of Goat Follistatin Gene CDNA

Follistatin is a single strand glycoprotein which is rich in cysteine.lt shows strong inhibit functions of FSH, so it is also called FSH-Suppressing Protein.Follistatin has several forms, its mRNA was expressed on different kinds of tissues and organs in the animals.As an activin-binding protein, follistatin involves in the animals reproductive regulating processes through the follistatin/activin system.There has not release Capra hircus follistatin cDNA sequence in public, so far.This study was research with 7 Dazu black goats,7 Nanjiang brown goats and 7 Saanen dairy goats as research object.Based on the Ovis aries(M63123.1)and Bos taurus(NM₁75801.2)follistatin cDNA released at the GenBank,the special primers of goat has been designed. The full length cDNA of goat was amplificated by the RT-PCR technique using the total RNA isolated from goat ovary as template. Then its potential amino acid sequence was deduced and its protein structure was analysed.Meanwhile,the tissue expression from different goat tissues was studied by RT-PCR and fluorescence quantitative PCR methods. The main results are as following:1,The full-length cDNA of goat follistatin gene was obtained from Dazu black goats, Nanjiang brown goats and Saanen dairy goats ovaries as template (GenBank accession number: HM138664～HM138666).Its length was 1217 nucletides, including 25 nucletides of 5'UTR and 158 nucletides of 3'UTR.The open reading frame (ORF) has 1035 nucletides that encoded a polypeptide of 344 amino acids, included 29 amino acids signal peptides and 1006 amino acids mature peptides.The homologies of the sequences were share 93% homology compared with the sequences of other mammals in GenBank. 2,The relative molecular weight of follistatin genes in Dazu black goats, Nanjiang brown goats and Saanen dairy goat were 37.942 KDa,37.801 KDa and 37.900 KDa, respectively. The isoelectric points were 5.85,6.07 and 6.07, respectively, the minimum values of hydrophobicity were -3.500,-3.500 and -3.156, respectively,the maximum were 3.511 of all. They had not protein transmembrane domains and had alpha helix (16.57%,15.99% and 20.06%), beta turn (4.94%, 4.65% and 2.91%), extended strand (17.15%,18.02% and 15.12%) and random coil (61.34%, 61.34% and 61.92%) in its secondary structure.3,Comparing the coding sequence of goats with other mammals,the cDNA sequence in goat had 3 mutation sites (including 2 transitionsal sites and 1 transversional sites) and 2 amino acids mutation sites. The cDNA sequence in Dazu Black goat had 2 mutation sites and two amino acids mutation sites.One of the mutation sites was 1025 (C→T) which was meaningless mutation.The other was 700 T→G that resulted in amino acid change (V→G). The cDNA sequence in Nanjiang brown goat has 1 mutation site 613 (A→G), that resulted in one amino acid change (E→G).4,The expression of follistin gene in different tissues was analyzed with Real-time PCR,the results showed that the follistatin gene was expressed in spleen, lung, kidney, liver, heart, ovary and pituitary. The expression level of follistatin gene in same varieties goat with different tissues were different. No significant difference was found in the spleen kidney and pituitary of different varieties (P>0.05). The expression level of follistatin gene in the lung liver and heart was extremely significant difference (P<0.01), and in ovary were significant differenc (P<0.05).5,Expression level of follistatin gene in every tissue of Saanen dairy goat was the highest in three goat breeds. Nanjiang brown goat was in middle level and Dazu black goat was in the lowest level.Saanen Dairy goat and Nanjiang Brown goat were significantly higher than. Dazu Black goat., (P<0.05). No significant difference was found between Saanen Dairy goat and Nanjiang Brown goat about the expression level of follistatin gene(P>0.05).Conclusion:The expression level of follistatin gene in goats was negative correlation with producing cade number, the high reproduction rate. was related to the low level of follistatin gene.