山羊MT-Ⅲ分子特性研究

通过设计特异性引物MT-ⅢSP1和MT-ⅢSP2,采用RT-PCR方法分别从奶山羊和绒山羊脑组织中克隆出金属硫蛋白-Ⅲ(MT-Ⅲ)基因编码区全序列,并利用生物信息学方法分析山羊MT-Ⅲ的分子特性.结果表明:奶山羊和绒山羊MT-Ⅲ基因编码区全长均为207bp,编码68个氨基酸,其中含19个半胱氨酸,不含芳香族氨基酸和组氨酸;BLAST搜索结果表明,MT-Ⅲ编码氨基酸序列在物种间高度保守,含有MT-Ⅲ特有的MDPET、C-X-C、C-C-X-C-C、C-X-X-C、KKS(X为非Cys外的其它氨基酸)等保守的短肽结构;生物信息学分析表明,山羊MT-Ⅲ无明显疏水结构和跨膜结构域,无信号肽,是一种细胞质蛋白.山羊MT-Ⅲ蛋白质二级结构主要为不规则卷曲,仅在第40-50位氨基酸残基间有明显的螺旋结构;三级结构预测表明,山羊MT-Ⅲ的三级结构由α和β2个结构域组成,通过KKS连接,且MT-Ⅲ氨基酸序列中第30位保守的半胱氨酸在反刍动物中均突变成丝氨酸,位于β-结构域.

Characteristics of metallothionein-Ⅲ in goat

A pair of special primers(MT-Ⅲ_(SP1) and MT-Ⅲ_(SP2)) were designed for cloning the coding sequence of MT-Ⅲ from brain tissue in goat. The MT-Ⅲ coding sequence in two kinds of goat were 207 bp. The two goat MT-Ⅲ gene sequences were submitted to GenBank under accession numbers EF471976 and EF195236. Both milkgoat and cashmeregoat MT-Ⅲ gene coded 68 amino acids, including 19 Cys residues.Goat MT-Ⅲ had characteristic MDPET, C-X-C.C-C-X-C-C,C-X-X-C and KKS sequences of MTs, where X was non-cysteine amino acids. The protein had no aromatic amino acids, no cross membrane area and no signal peptide, these information showed that MT-Ⅲ in goat was a cytoplamic protein . The second structure of goat MT-Ⅲ was random coil, except alpha helix structure in 40-50 amino acids. The tertiary dimension structure was composed by α and β-domain, the β-domain structure was same with the other species, but α- domain structure in goat was different with sheep and other species, because it had less one Cys in goat than the other animal. The structure change might alter the biological function of animal.