X染色体60149273位点在脂尾(臀)和瘦尾绵羊品种中的多态性及其基因定位

Polymorphism of the 60149273th Loci on X Chromosome Among Fat Tail and Thin Tail Breeds and Its Gene Mapping

【目的】研究绵羊X染色体60149273位点在不同沉脂尾型绵羊品种中的多态性,为解析绵羊尾脂沉积性状的遗传机制提供基础数据。【方法】首先,采用PCR-SSCP分型方法检测X染色体60149273位点在不同脂尾类型绵羊品种的多态性,并借助PCR产物直接测序的方法确认各基因型相应序列,利用卡方检验的方法检测各基因型分布在不同品种中的平衡性。其次,利用生物信息方法将该SNP定位与绵羊androgen receptor(AR),并使用电子克隆的方法克隆出绵羊AR全长编码序列。同时以阿勒泰羊臀部脂肪RNA为材料,采用RT-PCR的方克隆绵羊AR第3—8外显子序列,利用生物信息学方法分析序列同源性以及序列的物种进化保守性。【结果】①PCR-SSCP结果显示,在脂尾(臀)型绵羊品种阿勒泰羊和湖羊群体中X染色体60149273位点未检出多态性,GG基因型占100%;而瘦尾(臀)型绵羊品种中国美利奴和萨福克羊群体中则出现了多态性,GG基因型分别下降至10%和21%。②利用比较基因组学首次电子克隆出该SNP所属基因AR的全长编码区,并从阿勒泰羊臀脂mRNA中成功扩增羊源AR第3—8外显子片段,测序结果与电子克隆序列完全一致。③物种同源性比对进一步显示,AR在哺乳类动物中高度保守,哺乳动物各物种间同源性高达90%;进化树分析结果显示,牛、绵羊、海豚和猪等哺乳动物亲缘较近。【结论】首次发现绵羊AR第3内含子一处SNP在脂尾(臀)与瘦尾绵羊品种中存在较大差异,该SNP将可作为一个分子标记运用于低脂绵羊新品种的培育,同时该SNP所属AR也可作为一个重要候选功能基因应用于绵羊尾(臀)脂沉积分子机制的相关研究。

[Objective] The objective of this paper is to study the polymorphism of the SNP located at the position of 60149247 on X chromosome between fat tail （rump） and thin tail breeds, and to provide the basic data for analysis of the mechanism of tail fat deposition in sheep. [Method] Firstly, PCR-SSCP genotyping method was used to analyze genotypes among different tail types of sheep breeds, and definite allelic genes were cleared by PCR-direct sequencing, and the chi-square test was employed to detect the balance of genotype distribution in different breeds. Secondly, the SNP was mapped on sheep androgen receptor （AR） and the whole CDS sequences of AR were obtained by comparative genome and silicon cloning methods. Taking cDNA of Altay tail fat as DNA template, RT-PCR method was used to clone sheep AR spinning six exons, and sequence homology and phylogenetic tree were analyzed by bioinformatics methods. [Result]The results of PCR-SSCP showed that there is no polymorphism in fat-tail sheep breeds （Hu sheep and Altay sheep）, and the genotypes of the two breeds are all GG genotype. And there are three genotypes （GG, GAand AA） in thin-tail sheep populations （Chinese Merino sheep and Suffolk sheep）, and the frequency of GG genotype dropped to 10% and 21%, respectively. The whole CDS sequences of sheep AR were cloned by silicon cloning, and sheep AR containing the fragment from the 3rd exon to 8th exon of AR was successfully cloned in total RNA of Altay tail fat for the first time, and the sequences are completely the same to the sequences predicted by silicon cloning. The sequence alignment also showed that AR is highly conserved in mammalian animals, and the homology was up to 90% among different mammalian animals, and the phylogenetic tree analysis showed that sheep is relatively close to bovine, pigs and dolphin. [Conclusion] This study was the first to identify a novel SNP in the 3rd intron of the sheep AR associated with the trait of fat tail, and it hinted that the SNP will be used as a practical application of molecular markers in low-fat sheep breeding. At the same time, AR will also serve as an important candidate gene in studying tail fat deposition in Sheep.