三个猪品种Myf5基因的克隆及序列分析

Cloning and Sequence Analysis of Myf5 Gene in Three Pig Breeds

为明确猪Myf5基因的蛋白结构及功能位点,从香猪、大白猪和大白×长白杂交猪背最长肌提取总RNA,采用RT-PCR技术克隆Myf5基因,其cDNA长893 bp,包括完整的开放阅读框768 bp,编码255个氨基酸。将所测序列与GenBank上已知猪种序列进行相似性比较,结果表明:香猪Myf5基因中有3个碱基变异,其中2个突变不改变编码的氨基酸为无义突变,1个突变使其编码的氨基酸由甲硫氨酸(M)变为亮氨酸(L)。大白猪没有碱基变化;大白×长白杂交猪Myf5基因中的1个突变,使其编码氨基酸由甘氨酸(E)变为谷氨酸(G)。将香猪与其他物种进行核苷酸和氨基酸编码区序列的相似性分析及生物信息学分析。相似性分析表明,香猪Myf5基因氨基酸编码区序列与绵羊、牛、马、兔、黑猩猩、非洲象和小鼠的相似性较高,均在88%以上,推测Myf5基因在哺乳动物具有一定的保守性;Myf5蛋白二级结构以无规则卷曲为主,其次为α螺旋,偶有β转角。预测Myf5蛋白不含信号肽序列,有29个磷酸化位点。

To elucidate the protein structure and function site of Myf5 gene of pig, three myogenic regulatory factors 5 （Myf5） gene were separately cloned from the longissimus dorsi of Xiang pig, Large x White pig and Large White Landrace hybrid pig using RT-PCR strategy. The cDNA of Mff5 genes was 893 bp in length, in-cluding complete open reading frame （ORF） of 768 bp encoding 255 amino acids. Compared with the other pigs in the GenBank, Myf5 gene of Xiang pig has two nonsense mutations and one genetic variation which leads to amino acid conversion from methionine （M） to leucine （L）. In Large pig breed, the nucleotide se- quences of Myf5 gene were conserved, however, a mutation in Myf5 gene of White x Landrace hybrid breed leads to an amino acid alteration from glycine to glutamic acid. Based on bioinformatic analysis on the de-duced amino-acid sequences, Myf5 gene of the Xiang pig showed higher than 88% sequence similarity with that of Ovis aries, Bos taurus, Equus caballus, Oryctolaguz cuniculus, Pongo abelii, Loxodonta africana and Mus musculus, indicating a conserved sequence among mammals. The secondary structure of the encoded Myf5 protein is mainly the random crimp, followed by the a-helix, and rare β-turn. In Myf5 protein of pig, no signal peptide was found, however, 29 phosphorylation sites were investigated.