山羊Dkk1基因的结构、启动子区的多态性及其与生产性状的关联分析

Genomic Structure,Polymorphism in Promoter Region and Their Associations with Production Traits of Dkk1 Gene in Goats

本研究拟克隆山羊的Dkk1基因并对其多态性及其与山羊产绒量的关系进行分析,寻找与山羊产绒量有关的DNA标记。山羊Dkk1基因的获得采用同源克隆的方法,根据产绒量的高低混成2个基因组DNA池,PCR扩增测序寻找差异的核苷酸,然后采用PCR-SSCP方法检测Dkk1基因的多态性。结果,获得了山羊Dkk1基因3491bp的序列,由4个外显子和3个内含子组成,编码265个氨基酸残基,与其它哺乳动物的对应序列有较高的同源性。共发现33处单核苷酸替代和一处内含子2中四碱基的插入。对启动子区2个邻近的多态性位点(T593C-C603A)的分析表明,在所检测的5个山羊群体中,A均为优势等位基因,在该位点均处于Hardy-Weinberg平衡状态(P>0.05);不同基因型的白绒山羊个体在产绒量、单位体表面积产绒量、初生体质量、出生到断奶体增质量上差异不显著(P>0.1),但A等位基因(T593-603C)对产绒量较为有利。结果表明,山羊Dkk1基因与其它哺乳动物相应序列高度同源,非编码区具有较高的多态性,启动子区的2个多态性与绒山羊的生产性状无显著关联。

The objective of this study was to clone goat Dkk1 gene,identify its polymorphisms and find DNA markers associated with cashmere production.To clone goat Dkk1 gene,PCR with primers designed within conserved encoding region was performed.Polymorphisms were screened by PCR amplification and sequencing with two genomic DNA pools of goats differing in cashmere production,and detected by PCR-SSCP approach.A fragment of 3 491 bp in size for goat Dkk1 gene was amplified and sequenced,including four exons and three introns and encoding 265 amino acid residues.It has high homology with the corresponding sequences of other mammalian species both at mRNA and amino acid levels.Altogether 33 single nucleotide substitutions and an insertion of 4 bp in intron 2 were revealed.Two polymorphic sites in（T593C and C603A） the promoter region were detected in five goat populations.Allele A was predominant in all of the five goat populations,and all populations were in Hardy-Weinberg equlibrium（P0.05） at the two sites.The cashmere production,cashmere production per body surface area,birth weight,gaining weight from birth to weanling among different genotypes had no significant difference in White Cashmere goats（P0.1）,although allele A had a trend to increase cashmere production.Goat Dkk1 is homologous to other mammal species.Most of the polymorphisms were in noncoding region of goat Dkk1 gene.No significant association was found between the T593C and C603A polymorphisms in Dkk1 promoter region and cashmere production.