奶牛Klf4基因CDS区和3′UTR的克隆及生物信息学分析

Cloning and bioinformatics analysis of the CDS region and 3’UTR of Klf4 gene in dairy cow

以奶牛乳腺组织cDNA为模板,采用PCR技术扩增奶牛Klf4基因CDS区和3′UTR,同时构建PMIR-Klf4-3′UTR表达质粒;应用生物信息学方法对Klf4基因CDS区及其编码蛋白的理化性质、亚细胞定位、翻译后的修饰结构和蛋白结构进行生物信息学分析.结果表明:奶牛Klf4基因CDS区长度为1434 bp,可编码477个氨基酸,分子质量为50759.17 u,理论等电点为8.76,属于亲水性蛋白.Klf4基因3′UTR长度为366 bp,酶切重组PMIR-Klf4-3′UTR表达质粒得到长度分别为366和6470 bp的两条带,表明成功构建了重组质粒.同源性和遗传进化分析可知,牛Klf4基因与山羊、绵羊的亲缘性最近,达到98%以上,与斑马鱼的亲缘关系最远.KLF4蛋白有56个磷酸化位点,包含16个苏氨酸磷酸化位点、37个丝氨酸磷酸化位点和3个酪氨酸磷酸化位点.在二级结构和三级结构中,KLF4蛋白α-螺旋占18.03%,无规则卷曲占68.76%,存在3个锌指结构域.本研究为进一步探讨Klf4基因在奶牛乳腺炎表观遗传学调控过程中的作用提供数据支撑.

cDNA from cow mammary gland tissue was used as a template to amplify the coding sequence(CDS)region and 3’untranslated region(UTR)of the Klf4 gene by PCR,then the PMIR-Klf4-3’UTR expression plasmid was constructed.Subsequently,the Klf4 gene CDS,protein physicochemical properties,subcellular localization,post-translational modification structure and protein structure were predicted and analyzed by bioinformatics software.The results showed that the CDS of cow Klf4 gene was 1434 bp in length,encoding 477 amino acids.Its molecular weight was 50759.17 u and it was a hydrophilic protein,with theoretical isoelectric point being 8.76.The length of Klf4 gene 3’UTR was 366 bp.Two bands of 366 bp and 6470 bp were digested by the recombinant PMIR-REPORT-3’UTR expression plasmid,indicating that the recombinant plasmid was successfully constructed.Homology analysis and genetic evolution analysis revealed that cow Klf4 gene was most related to goat and sheep,reaching more than 98%,while it was least related to zebrafish.KLF4 protein had 56 phosphorylation sites,including 16 threonine phosphorylation sites,37 serine phosphorylation sites,and 3 tyrosine phosphorylation sites.In the secondary structure and tertiary structure,theα-helix of KLF4 protein accounted for 18.03%,and the random coil accounted for 68.76%.There were 3 zinc finger domains in KLF4 protein structure.This study is contributed to further research on the role of Klf4 gene in the epigenetic regulation of dairy cow mastitis.