天祝白牦牛金属硫蛋白基因的克隆及表达

Cloning and expression analysis of metallothionein-2(MT-2)gene in Tianzhu white yak

运用RT-PCR和RACE(rapid amplification of cDNA ends,RACE)技术获得天祝白牦牛金属硫蛋白(MT-2)基因全长cDNA序列,对其生物学进行分析,并通过荧光定量PCR双标准曲线法和2-ΔΔct法建立检测MT-2基因表达的方法.结果表明:扩增获得的天祝白牦牛MT-2基因全长为410bp(GenBank登录号:KF888633),ORF长185bp,编码61个氨基酸.该基因无跨膜结构,为非分泌型蛋白.构建系统进化树结果显示,天祝白牦牛MT-2基因在不同物种以及进化的过程中具有高度保守性,与黄牛和羊的亲缘关系最近.荧光定量PCR结果表明,双标准曲线法特异性强、灵敏度高、稳定性好,对天祝白牦牛MT-2的定量检测具有重要意义.

Full length cDNA sequence of MT-2 gene in Tianzhu white yak was cloned by RT-PCR and RACE （rapid amplification of cDNA ends,RACE）.Double standard curves and 2-delta Ct methods were applied to develop a sutitable qRT-PCR method to analysis the expression of MT-2.The results indicated that the full length of MT-2 cDNA sequence was 410 bp （Gene Bank accession No.KF888633）,with an ORF 185 bp in length,which encoded 61 amino acids.MT-2 was a non-secreted protein without transmem-brane structure.Phyletic evolution analysis suggested that the highest homology was appeared with Bos taurus,and was highly conserved in different species and the process of evolution.qRT-PCR demonstrated that double standard curves method with strong specificity,high sensitivity and good stability,had an im-portant meaning for the quantitative detection of MT-2 .