绵羊MC4R基因的半定量RT-PCR及生物信息学分析

Semi-quantitative RT-PCR and Bioinformatics Analysis of Sheep MC4R Gene

本研究旨在对绵羊MC4R基因进行组织表达谱和生物信息学分析。参考牛MC4R基因序列设计引物,采用PCR技术克隆绵羊MC4R基因序列,并利用半定量RT-PCR进行组织表达谱分析;同时对其进行生物信息学分析。结果表明,克隆的绵羊MC4R基因全长1 919 bp,含有999 bp的完整CDS编码区,编码332个氨基酸。其CDS编码区的核苷酸序列与牛、人、猪、大鼠、小鼠MC4R基因对应序列的同源性分别为95.2%、68.8%、82.7%、76.6%、77.1%,预测的氨基酸序列同源性分别为97.0%、92.8%、93.7%、92.2%、91.6%。组织表达谱分析表明MC4R基因在各组织均不同程度的表达,其中在大脑表达量很高,其他组织较低。生物信息学预测MC4R蛋白功能发现,绵羊的MC4R蛋白存在7个跨膜螺旋结构域,同时预测MC4R存在10个磷酸化位点和1个特异性蛋白激酶磷酸化位点。结果表明,MC4R是一个非常保守的蛋白,在绵羊的生长发育中起着重要作用。

The aim of this study was to analyze the tissue-specific expression profile and bioinformatics characteristic of MC4R in sheep.Primers were designed based on the sequence of bovine MC4R gene and were used to amplify the sheep MC4R gene by PCR,the tissue-specific expression profile was analyzed by semi-quantitative RT-PCR,and the bioinformatics analysis of MC4R was conducted.The results showed that CDS sequence of sheep MC4R was 999 bp,encoding a deduced protein containing 332 amino acid residues,the full length of MC4R gene cloned was 1 919 bp,and this nucleotide sequence of CDS shared 95.2%,68.8%,82.7%,76.6% and 77.1% homology with the MC4R mRNA of cattle,human,pig,rat and mouse,while the homology of deduced amino acids were 97.0%,92.8%,93.7%,92.2%,91.6%,respectively.Semi-quantitative PCR revealed that the MC4R gene was expressed in various tissues,but at different levels.The expression level of this gene was higher in the brain,and lower in other tissues.It was predicted that MC4R was a kind of transmembrance protein and had seven transmembrane domains in sheep.There were ten phosphorylation sites and one phosphorylation site of specific protein kinase in MC4R protein.The results indicated that MC4R was an evolutionarily conserved protein,and played an important role in growth of sheep.