【作者】 王全喜；

【导师】 芒来； 张立岭；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 肌肉生长抑制素(Myostatin,MSTN)是近年来发现的一种生长分化因子,主要在动物骨骼肌中表达,它对动物肌肉的发生和发育以及再生都有重要调控作用。该基因缺失会导致动物骨骼肌肌纤维数量增多或肌细胞肥大等现象,这对肉用养马业和速度赛马业有潜在的应用价值。但国内外对马MSTN基因的研究较少,未曾见到马的MSTN基因全序列的报道,更未见对马的MSTN基因的生物信息学研究和基因多态性分析报道。因此,本研究从蒙古马的MSTN基因的克隆和全序列测序入手,进一步对马的MSTN基因进行生物信息学分析研究,并对乌审马、乌珠穆沁马、锡尼河马、三河马、纯血马、矮马等六个品种马MSTN基因编码序列进行多态性分析,目的是为建立蒙古马基因文库提供可靠信息,并为马MSTN的分子生物学研究及分子育种奠定理论基础。本研究取得了以下结果: 首先克隆了蒙古马MSTN基因并进行了全序列测定,测定结果登录在Genbank上,登录号为AY840554,全序列分析结果表明,蒙古马MSTN基因全序列为4979bp,由三个外显子和两个内含子组成,即第一外显子373bp、第二外显子374bp、第三个外显子381bp、第一内含子1833bp、第二内含子2018bp。cDNA全长为1128bp,只出现一个开放的读码框,因此可以推测马MSTN基因蛋白前体由375个氨基酸组成。 其次,借助生物信息软件,对蒙古马MSTN基因进行序列对比以及推测编码蛋白质的一、二级结构和一些理化性质:①序列对比结果表明,马MSTN基因序列与GenBank中的马AB033541序列基本一致,cDNA序列同源性达到99.56%,只有5个碱基的差异,而且其中2个碱基的突变为沉默突变;氨基酸序列同源性达到99.21%,只有3个氨基酸的差异。②氨基酸序列与其它动物同源序列对比结果表明,所推测出的氨基酸序列具有TGF-β超家族成员结构特点。④MSTN基因分子进化分析结果表明:马与猪的亲缘关系最近,与牛、羊、狗、狐狸、鼠的亲缘关系较近,其次是与鹅、火鸡、鸡和鹌鹑等鸟类的亲缘关系,与鱼类的亲缘关系较远。⑤蛋白质理化性质推测结果表明:此基因编码的蛋白质分子量为42.9kd,等电点为7.59的大分子。⑥最后蛋白质二级结构预测表明,该蛋白质是含有84个α螺旋、77个β折叠和214个无规卷曲的多肽。 再次,对上述六个品种马的MSTN基因的三个外显子进行PCR-SSCP分析,并计算出遗传参数,进行了群体遗传学特性分析:结果表明,第一和第三外显子出现多态,第二外显子中未检测到SNPs位点,经X~2检验发现,第一外显子的突变在六个品种马均处于平衡状态,第三外显子的突变在乌审马和纯血马群体中处于不平衡状态,其余品种中均处于平衡状态。基因杂合度、有效等位基因数以及多态信息含量等的计算发现,6个品种马的突变位点PIC表现均为中度多态。更多还原

【Abstract】 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.更多还原