牦牛MSTN基因分子克隆及序列分析

Cloning and Sequence Analysis of Yak MSTNGene

设计特定引物对牦牛MSTN基因PCR分段扩增并克隆和测序,利用分子生物学软件进行序列拼接,获得牦牛MSTN基因序列(GenBank登录号EU926670)。该基因由3个外显子和2个内含子组成,CDS序列全长为1 128 bp(GenBank登录号EU926671),由375个氨基酸组成。外显子大小分别为373,374,381 bp,内含子大小分别为1 843,2 028 bp。牦牛与普通牛的MSTN基因编码区中,在417位发生一次碱基转换(C→T),但未造成氨基酸改变。不同物种间在该基因编码区核苷酸序列和氨基酸序列上有较高的相似性,牦牛与普通牛、绵羊、猪、人、狗、小鼠、马、兔子、鸡、猩猩各物种间核苷酸相似性大小分别为99.9%,96.5%,94.3%,89.1%,91.9%,91.3%,93.6%,91.7%,82.0%,92.0%。牦牛与普通牛MSTN氨基酸序列相似性最高,为100%;而与绵羊、猪、小鼠、人、狗、马、兔子、鸡、猩猩各物种间相似性大小分别为93.3%,95.5%,92.5%,94.1%,93.3%,94.9%,94.4%,88.0%,94.4%。生物信息学软件分析发现:牦牛MSTN基因编码蛋白的理论分子量约为42.6 kDa,PI值为6.14,Leu的含量最高(9.9%),其次是Lys(7.2%)。牦牛MSTN基因编码蛋白二级结构以β折叠为主,属于跨膜蛋白,跨膜区位于AA6-AA23之间;具有一个分泌信号肽结构,其氨基酸序列MQKLQICVYIYLFMLIVA具有TGF-β家族的特征。

The yak MSTN gene was amplified using particular primers,then cloned and sequenced.It was obtained by sequence splicing using molecular biology software.The results showed that yak MSTN gene（GenBank Accession No：EU926670）had 3 exons and 2 introns.The size of CDS（GenBank Accession No：EU926671）sequence of the yak MSTN gene,encoding 375 amino acid,was 1 128 bp.The size of exonⅠ,exonⅡand exon Ⅲ was 373,374,374 bp,respectively;while the size of intron Ⅰ and intronⅡwas 1 843,2 024 bp,respectively.A single base conversion（C→T）located 417 base was found in Yak MSTN gene coding region compared to ordinary cattle,which did not cause changes in amino acids.The nucleotide sequence of the coding region and deduced amino acid sequences among different species were quitely conservative.The homology rates of nucleotide sequences of the coding region of MSTN gene between the yak and cattle,sheep,pig,human,dog,mouse,house,rabbit,chick and chimpanzee were 99.9%,96.4%,94.6%,92.2%,91.8%,91.8%,92.8%,95.3%,84.4%,84.9%,respectively,while the homology rates of amino acid sequences were 100%,93.3%,95.5%,92.5%,94.1%,93.3%,94.9%,94.4%,88.0% and 94.4%,respectively.Biological software analysis showed that the theoretical molecular weight of Yak MSTN gene was 42.6 kDa and PI was 6.14.Leu was the highest（9.9%）,followed by Lys（7.2%）.The secondary structure of Yak MSTN protein was mainly β-folding and it had characteristics of transmembrane protein structure.And the transmembrane domain located between AA6 and AA23.Moreover,it had a secretion signal peptide structure,MQKLQICVYIYLFMLIVA,which probably related well with TGF-β protein family.