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Cloning and SNPs analysis of MC1R gene of hybrid F2 generation of Landace × Bama minipig

  • 摘要: 【目的】探讨猪黑素皮质激素受体Ⅰ基因(MC1R)碱基突变与其毛色分离的相关性,为研究猪的毛色遗传分子机制及遗传育种奠定基础。【方法】根据GenBank已公布的猪MC1R基因同源序列(AF326520)设计引物,以70头不同毛色的长白猪×巴马小型猪杂交F2代(长×巴F2代)猪血液总DNA为模板,PCR扩增MC1R基因序列,并利用PCR-SSCP对长×巴F2代杂交猪的MC1R基因进行单核苷酸多态性(SNP)分析。【结果】长×巴F2代杂交猪MC1R基因编码区序列约963 bp,与参考序列(AF326520)的同源性为99.37%,共有6处碱基发生突变,其中两处(284G→A和306T→C)为错义突变,284G→A导致95Val→Met,306T→C导致102Leu→Pro;其余4处均为同义突变,分别为6C→T、51G→A、364T→C和730G→A。 PCR-SSCP检测结果显示,长×巴F2代杂交猪的MC1R基因均未表现出多态性。【结论】长×巴F2杂交猪的MC1R基因存在碱基突变,其基因型为ED1,但在不同毛色的长×巴F2代杂交猪群体中并未发现MC1R基因呈多态性,即猪毛色多样性不能简单以MC1R基因的多态性进行分析。

     

    Abstract: ObjectiveThe influence of base mutation of melanocortin Ⅰ receptor (MC1R) gene on segregation of coat color was studied to provide references for its breeding and genetic mechanism of coat color. MethodPrimers were designed according to the MC1R gene coding sequence ( AF326520 ) obtained from GenBank . The total DNA in blood of 70 hybrid F2 generation of Landace ×Bama minipig was used as the template for PCR. The coding sequence of MC1R was amplified and cloned by PCR. SNPs analysis of MC1R gene was performed using PCR-SSCP. ResultMC1R gene coding sequence (CDS) of the hybrid F2 generation of Landace and Bama minipig was 963 bp, in which similarity was 99.37%, compared with its reference sequence. MC1R gene coding sequences (CDS) in the hybrid F2 generation of Landace and Bama minipig had six mutations compared to sequences of MC1R gene from GenBank. The base 284G→A caused amino acid 95Val→Met and 306T→C led to 102Leu→Pro , except 4 synonymous mutations , which were 6C→T, 51G→A, 364T→C and 730G→A . PCR-SSCP analysis showed that MC1R displayed no polymorphism. Con-clusionMC1R gene has polymorphism in the hybrid F2 generation of Landace×Bama minipig and its genotype is ED1,but none of polymorphism is found from the CDS of MC1R gene in the hybrid F2 generation of Landace×Bama minipig, in which coat color was different , so coat color polymorphism can not be simply explained by polymorphism of MC1R.

     

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