利用PCR-SacⅡ-RFLP技术检测绵羊IGFBP-3基因多态性的研究

PCR-SacⅡ-RFLP Detecting Polymorphism at the Sheep IGFBP-3 Gene

【目的】将IGFBP-3基因作为候选基因,寻找与绵羊羊毛品质等经济性状相关的SNP位点。【方法】利用PCR-SSCP和DNA测序快速筛查SNP位点;在此基础上建立PCR-RFLP检测方法,分析该位点在不同品种绵羊中的基因型和等位基因分布情况;通过多重比较分析不同基因型与中国美利奴羊部分羊毛性状的关联性。【结果】以绵羊基因组DNA为模板扩增得到了249 bp的特异条带,PCR-SSCP及测序分析显示在该序列的140碱基处发生了G/T突变,导致SacⅡ酶切位点消失。经PCR-SacⅡ-RFLP检测,哈萨克羊和中国美利奴羊细毛羊得到三种基因型:TT(249 bp)、GG(139 bp/110 bp)和TG(249 bp/139 bp/110 bp);杜泊羊中出现GG和TG两种基因型;湖羊、小尾寒羊以及萨福克羊中只有GG一种基因型。关联性分析表明不同基因型与部分羊毛性状没有明显的相关性。【结论】在绵羊IGFBP-3基因嫩含子4区发现一个新的SNP位点,玻建立PCR-SacⅡ-RFLP检测方法;该位点不同等位基因和基因型在不同绵羊品种中的分布具有一定规律性;不同基因型与中国美利奴细毛羊部分羊毛性状中间没有明显的相关性。

【Objective】To seek the SNP associated with the economic traits such as wool traits in sheep,IGFBP-3 regarded was as candidate gene reflecting growth and development.【Method】The polymorphism of sheep IGFBP-3 gene was first detected by PCR-SSCP and DNA sequencing.On the basis,the PCR-RFLP method for the special SNP was established and used to identify the genotype and allele frequencies in different sheep breeds（native breeds HU sheep,Small-tail Han sheep and Kazakh sheep,breeding variety Chinese Merino sheep,foreign breed Suffolk sheep and Dorper sheep）.The association between different genotypes and several wool traits of Chinese Merino sheep was analyzed by multi-comparison method.【Result】An expected PCR product of 249 bp was amplified using the sheep genomic DNA as temple.PCR-SSCP and sequence analysis revealed a G/T transversion at position 140 of the fragment,resulting in the SacⅡ site disappeared.Digested by SacⅡ to the PCR product,Kazakh sheep and Chinese Merino sheep illustrated three unique binding genotypes： 249 bp（TT）、139 bp/ 110 bp（GG） and 249 bp/139 bp/110 bp（TG）;Dorper sheep had two genotypes：139 bp/110 bp（GG） and 249 bp/139 bp/110 bp（TG）;Only GG genotype was detected in HU sheep,Small-tail Han sheep and Suffolk sheep. 【Conclusion】A novel SNP was found in the Intron 4 of sheep IGFBP-3 gene and the PCR-SacⅡ-RFLP method for the site was established.The distribution of genotype and allelic frequencies at this site differed among the studied sheep breeds;There were not significant association between different genotypes and several wool traits of Chinese Merino sheep.