牦牛MT-IV基因克隆与序列分析

Molecular cloning and bioinformatics analysis of MT-IV in the yak

采用RT-PCR方法,利用特异性引物YMT-IVSP1和YMT-IVSP2先克隆出牦牛Bos grunniens MT-IV的编码区序列全长,将其与人MT-IV基因全序列比对,设计2对引物分别克隆内含子1和内含子2,将获得序列拼接后得到了牦牛MT-IV外显子与内含子全长为2 099 bp(GenBank Accession No.:EU665491),编码区序列长189 bp,编码62个氨基酸,20个半胱氨酸残基,不含芳香族氨基酸,2个内含子的长度分别为1 392和518 bp。将牦牛MT-IV基因编码区序列和氨基酸序列BLAST搜索结果表明,MT-IV在物种间高度保守。牦牛MT-IV氨基酸序列与牛、绵羊、山羊、狗、家鼠、人和马的比对表明,MT-IV包含有金属硫蛋白(MTs)特有的C-X-C、C-C-X-C-C、C-X-X-C结构域,构建的系统发育树与比较生理学和形态学相同,表明牦牛MT-IV具有与其他哺乳动物相同的分子特性,有必要进行牦牛MT-IV在上皮细胞中表达量水平的研究。

In this ,study, coding sequence of yak metallothionein （MT--IV） gene was cloned by reverse transcriptase-polymerase chain reaction （RT--PCR） using the gene specific primers YMT-IVSP1 and YMT-IVSP2, and then 2 pairs specific primers were designed for cloning 2 introns of the yak MT--IV by comparing with the human MT--IV gene. The sequence length of MT--IV gene in yak was 2 099 bp （GenBank Accession No. ： EU665491）, including 3 exons and 2 introns. The coding regions were composed by 189 nucleotides encoding a 62-amino acid protein which contained 20 cysteine residues and no aromatic AA. The length of 2 introns were 1 392 bp and 518 bp, respectively. The MT--IV proteins were highly conserved among yak, cattle, sheep, goat, dog, mouse, human and horse, which had a glutamate insertion at position 5 relative to the classical MT-I and MT-I1 proteins, and also had the conserved motifs of MTs, such asC--X--C,C--C--X--C--C,C--X--X--C. These results indicated that the yak MT--IV had same molecular characteristics with other mammalian species, but it was necessary to study the expression level of the yak MT--IV in stratified squamous epitheliat.