绵羊GSTA2基因克隆、生物信息学分析及真核表达载体构建

Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of GSTA2 Gene in Sheep

为阐明绵羊谷胱甘肽S转移酶α2(Glutathione S-transferase alpha 2,GSTA2)基因功能,利用牛GSTA2基因序列设计引物,提取绵羊皮肤组织总RNA,应用RT-PCR、基因克隆、生物信息学对GSTA2基因进行研究,构建真核表达载体,转染绵羊皮肤成纤维细胞,应用qPCR技术检测GSTA2基因的表达。结果表明,克隆到绵羊GSTA2基因672 bp编码区序列(GenBank登录号:OR440604.1),编码223个氨基酸,分子量为25.39 ku。氨基酸比对和系统进化树分析表明,绵羊与牛的GSTA2相似度最高,达87.39%。GSTA2的二级结构以α-螺旋和无规则卷曲为主。构建的真核表达载体pcDNA3.1-GSTA2转染绵羊皮肤成纤维细胞后,GSTA2基因表达量极显著上调(P <0.001)。说明成功克隆了绵羊新基因GSTA2的CDS序列,并成功构建了其真核表达载体。

To study the function of glutathione S-transferase alpha 2(GSTA2)gene in sheep,primers were designed according to bovine GSTA2 gene sequence,and total RNA were extracted from sheep skin tissue.GSTA2 were cloned using RT-PCR and eukaryotic expression vector was constructed.The sequence of GSTA2 was analyzed using bioinformatics analysis.Sheep fibroblasts were transfected with GSTA2 eukaryotic expression vector,and the expression of GSTA2 mRNA was detected by qPCR technique.The results showed that CDS of sheep GSTA2 gene(GenBank accession number:OR440604.1)was cloned.The length of GSTA2 CDS was 672 bp,and encoded 223 amino acids.Its molecular weight was 25.39 ku.Amino acid comparison and phylogenetic tree analysis showed that sheep GSTA2 had the highest similarity to Bos taurus,reaching 87.39%.The secondary structure of GSTA2 was mainly composed ofα-helix and random coil.The recombinant expression vector pcDNA3.1-GSTA2 was constructed,and the expression level of GSTA2 gene was significantly up-regulated after transfected into sheep fibroblasts(P<0.001).It was indicated that a new gene(GSTA2)CDS of sheep was successfully cloned and its eukaryotic expression vector was correctly constructed.