绵羊角蛋白关联蛋白2基因的克隆表达与生物信息学分析

本研究旨在克隆新吉细毛羊和小尾寒羊的角蛋白关联蛋白2（keratin-associated protein 2,KAP2）基因并对其进行生物信息学分析,并初步探讨KAP2基因与羊毛毛用性状分子的关系。分别提取新吉细毛羊和小尾寒羊皮肤组织中总RNA,用RT-PCR方法扩增KAP2基因,将PCR产物克隆于pMD19-T载体,转入大肠杆菌DH5α感受态细胞,筛选阳性克隆。提取扩增后的重组质粒pMD19-T-KAP2并进行PCR扩增、双酶切鉴定、序列测定及蛋白质结构预测。结果表明,试验成功克隆了大小为463 bp的KAP2基因,开放阅读框架（ORF）长414 bp,编码137个氨基酸。测序结果比对发现,与小尾寒羊KAP2基因相比,新吉细毛羊的KAP2基因中存在1个由G→A的碱基突变,编码氨基酸由精氨酸（Arg）变为谷氨酰胺（Gln）,属于错义突变。新吉细毛羊和小尾寒羊的KAP2蛋白的分子质量分别为14.81和14.84 ku,等电点分别是9.67与9.82,两种绵羊KAP蛋白的基本理化性质无明显差异,同属于碱性疏水性、跨膜蛋白;预测到蛋白二级结构均由α螺旋、延伸链、无规则卷曲等构成,新吉细毛羊和小尾寒羊KAP2蛋白三级结构中存在一个由α螺旋引起的空间结构的差异位点。综合以上结果推测KAP2基因可能是影响羊毛性状的差异基因,本试验结果可为探讨两种绵羊毛用性状表型差异的分子遗传奠定理论基础。

Cloning,Expression and Bioinformatics Analysis of KAP2 Gene in Sheep

The keratin-associated protein 2 (KAP2) gene was cloned from Xinji Fine-wool sheep and Small-tail Han sheep,the KAP2 proteins was analyzed by bioinformatics,and the relationship between KAP2 gene and molecules trait of wool hair was preliminary analyzed in this study.The total RNA was extracted from wool follicles of Xinji Fine-wool sheep and Small-tail Han sheep.The KAP2 gene was cloned by RT-PCR,and the PCR product was cloned into pMD19-T vector,then was transferred to Escherichia coli DH5α competent cells and the requisite clones were filtered,and the recombinant plasmid was extracted.Identification of PCR and double enzyme digestion and sequencing were operated,then the prediction of protein structure was analyzed by bioinformatics.The results showed that the length of KAP2 gene was 463 bp,containing an ORF that was 414 bp and encoded 137 amino acid residues.The comparison of sequencing results showed that compared with the KAP2 gene in Small-tail Han sheep,there was a base mutation from G to A in KAP2 gene of Xinji Fine-wool sheep which was a missense mutation causing the encoded amino acid changed from arginine (Arg) to glutamine (Gln).The molecular weight of KAP2 protein in Xinji Fine-wool sheep and Small-tailed Han sheep were 14.81 and 14.84 ku,and the isoelectric points of KAP2 protein were 9.67 and 9.82,respectively.The KAP2 protein of the two breeds was no obvious differences in physical and chemical properties,which was transmembrane, alkaline and hydrophobic protein.It was predicted that the secondary structure of KAP2 protein was composed of α-helix,extend chain and random coil.The tertiary structure of KAP2 protein in Xinji Fine-wool sheep and Small-tail Han sheep had a different spatial structure that came from α-helix.Therefore,it could be speculated that KAP2 gene might be a differential gene affecting wool quality.The study of KAP2 gene provided a theoretical basis for exploring the molecular inheritance of phenotypic differences between two wool species.