山羊、绵羊MT-IV分子特性研究

Characteristics of MT-IV in sheep and goat

金属硫蛋白（MTs）是一类低分子量、金属和半胱氨酸含量高的细胞质蛋白，哺乳动物的MTs包括MT-I、MT-Ⅱ、MT-Ⅲ和MT-Ⅳ4种亚型，其中MT-Ⅳ只在磷状复层扁平上皮细胞中表达，相关研究报道较少。本研究根据GenBank已公布的动物MT-Ⅳ基因序列，设计出扩增山羊和绵羊MT-Ⅳ基因的特异性PCR引物MT-IVSP1和MT-ⅣSP2，利用RT-PCR的方法，分别从山羊和绵羊的瘤胃组织mRNA中，克隆出山羊和绵羊的MT-IV基因编码区序列（均为189bp），序列登录GenBank，获得序列号EF470251和EF624067。通过序列分析，表明山羊和绵羊两个物种MT-Ⅳ基因编码区全编码均为189bp、编码62个氨基酸，其中绵羊的MT-Ⅳ含有20个半胱氨酸，而山羊第6l位保守的半胱氨酸被色氨酸所代替。两个物种的MT-IV均不含芳香族氨基酸，含有MTs特有的C—X—C、C—X—X—C、C—C—X-C．C结构，无明显的跨膜结构域，无信号肽，是一种细胞质蛋白。二级结构分析表明两个物种的MT-Ⅳ二级结构大多数为无规则卷曲结构，分别在第7-9和第49～51氨基酸残基性存在折叠结构，不存在螺旋结构。三级结构预测结果表明两个物种MT-Ⅳ的三级结构由0c和D两个结构域组成，其中β结构域相同，仪结构域山羊少一个半胱氨酸残基，其结构与绵问存在明显差异，这一差异可能对山羊MT-Ⅳ的生理功能产生一定影响，有必要深入研究。

Metallothioneins （MTs） was characterized by its low molecular weight （6 to 7 kDa）, high metal content, high content of conserved cysteine（Cys） residues and absence of aromatic amino acids. In mammals there are four isoforms （MT-I-Ⅳ）, MT-Ⅳ was expressed exclusively in stratified squamous epithelia. In this study, according to other animal MT-Ⅳ gene sequence from GenBank, a pair of special primers （MT-/Vsp1 and MT-Ⅳsp2） were designed for cloning the coding sequence of MT-Ⅳ from rumen organ in both sheep and goat. The MT-Ⅳ coding sequence in goat and sheep were 189bp, and be submitted to GenBank under Accession Numbers EF470251 and EF624067, respectively. Both goat and sheep MT-Ⅳgene coded 62 AAs, including 20 Cys residues in sheep, 19 Cys residues in goat, the 61st conserved Cys was replaced by Trp in goat. MT-Ⅳ in goat and sheep both have characteristic Cys-x-Cys, Cys-x-y-Cys, and Cys-Cys sequences of MTs, where x and y were non-cysteine amino acids, no aromatic AAs, no cross membrane area, and no signal peptide, these information showed MT-Ⅳ in goat and sheep was a cytoplamic protein. The second structure of MT-Ⅳ was coil, except sheet structure in 7-9 AAs and 49-51AAs. The 3 dimension structure was composed by α-and β-domain, theN-domain structure was same between sheep and goat, and same with other species, but α- domain structure in goat was difference with sheep and other species, because it less one Cys in goat than other animals,the structure change maybe alter the biological function, it is necessary to study furtherly.