Cloning And Sequence Analysis Of Mongolian Horse MSTN Gene And Polymorphism Studies On Some Horse Breeds

Myostatin (MSTN) is a kind of newly discovered growth differentiation factor, mainly expressed in animal skeleton muscles, which is taking an important role in the growth and regeneration of animal muscles. The deficiency of this gene would cause the increasing of the quantity animal skeleton muscles fiber and becoming fleshy of muscle cells, there is latent using value on flesh horse and horse racing industry. The study about horse MSTN gene was rarely reported in the present reports, the all sequence of MSTN gene, bioinformatics research and polymorphism analysis was never reported before. In this thesis, full sequence of Mongolian horse MSTN gene was cloned and sequenced. The sequence structure, nucleotide composition and amino acid were analyzed by bioinformatics methods. Furthermore, full sequences of 6 breeds(Wushen hotse,Wuzhumqin horse,Xinihe horse,Sanhe horse,Thoroughbred horse and pony) were analyzed by PCR-SSCP. The results showed:The all sequence of Mongolian horse MSTN gene was cloned and sequenced and submitted in GenBank (accession AY840554).The result showed: the all sequence was 4979bp including 3 exons and 2 introns, the size of the exons were: 373bp,374bp,381bp;the size of the introns were:1833bp and 2018bp.The cDNA was 1128bp,there was only one opening frame ,it can be inferred that there was 375 amino acids in horse MSTN former protein.Secondly, the analysis of the sequence structure and primary, secondary structure of MSTN gene showed that:①Compared with horse MSTN gene which was submitted in GenBank (accession AB033541),the homologies of cDNA was 99.56%,there were only 5bp changed ,among them two were silence mutation. The homologies of amino acid were 99.21%; only three amino acids were different. ②The analysis of homologies of amino acid showed that the inferred amino acid sequence has the character of TGF-βfamily.③The molecular evolution trees of MSTN gene showed that the distance between pig, ovine, goat, dog were the nearest, between birds was nearer and between fish was the most far. ④The analysis of physics and chemical character of MSTN gene indicated that the protein's molecular quantity was 42.9KD,isoelectric point was 7.59. ⑤The analysis of secondary structure of protein showed that there were 84 a-helix,77 β-sheet and 214 irregular coiled peptide.The result of PCR-SSCP analysis for 3 exons of MSTN gene among the 6 horse breed indicate that there were polymorphism in exon 1 and exon3,there was no SNPs in exon2 site . Analyzed by X2 test, the mutation of exonl in 6 breeds were in balance condition ;the mutations of exon3 of Thoroughbred horse and Sanhe horse were not in balance condition, the others were in balance. They were in average polymorphism in PIC mutation site in 6 horse breeds by other analysis.