绵羊DIO2基因组织表达及其多态性与产羔数关联分析

Tissue expression and polymorphism of sheep DIO2 gene and their association with litter size

[目的]本试验旨在探明Ⅱ型脱碘酶(DIO2)基因在常年发情的小尾寒羊和季节性发情的草地型藏羊中的表达模式,并探讨其多态性与绵羊季节性繁殖和产羔数之间的关系。[方法]通过荧光定量PCR(qPCR)检测DIO 2基因在不同品种绵羊的10种组织中的表达,同时利用SNP技术对3个常年发情绵羊品种(小尾寒羊、策勒黑羊和湖羊)和3个季节性发情绵羊品种(滩羊、苏尼特羊和草地型藏羊)的DIO 2基因2个SNP位点多态性进行检测,并与小尾寒羊产羔数进行关联分析。[结果]qPCR结果显示,DIO2基因在小尾寒羊和草地型藏羊各组织中广泛表达,其中草地型藏羊下丘脑、垂体、卵巢、子宫和输卵管组织中的表达量显著高于小尾寒羊(P<0.05)。基因分型结果表明,绵羊DIO2基因g.88484603G>C位点基因型频率和等位基因频率在季节性发情和常年发情绵羊品种间差异均达到极显著水平(P<0.01)。群体遗传学分析表明,绵羊DIO 2基因g.88484594T>C位点在滩羊和策勒黑羊中表现为中度多态(0.25<PIC<0.5),在其他4个群体中均表现为低度多态(PIC<0.25);g.88484603G>C位点在这6个绵羊群体中均表现为中度多态(0.25<PIC<0.5)。卡方适合性检验结果表明,g.88484594T>C位点在这6个群体中均处于哈代温伯格不平衡状态(P<0.05),g.88484603G>C位点在这6个绵羊品种中均处于哈代温伯格平衡状态(P>0.05)。生物信息学分析表明,g.88484603G>C位点突变前、后的mRNA二级结构发生了改变,而且其最小结构自由能降低,结构稳定性增强,蛋白质二级结构没有发生变化,但三级结构有一些变化。关联分析表明,这2个SNP位点与小尾寒羊前3胎产羔数均无显著关联(P>0.05)。[结论]DIO 2基因与绵羊的季节性繁殖密切相关,其g.88484603G>C位点对绵羊季节性繁殖性状有一定的调控作用。

[Objectives]The paper aimed to study the expression pattern of TypeⅡthyroid hormone deiodinase(DIO2)gene in year-round estrous Small Tail Han sheep and seasonal estrous Prairie Tibetan sheep,and to explore the relationship between the polymorphism and the seasonal breeding and the litter size in sheep.[Methods]qPCR was performed to investigate the expression of DIO 2 gene in 10 tissues at different breeds of sheep.Year-round estrous(Small Tail Han sheep,Cele Black sheep and Hu sheep)and seasonal estrous(Tan sheep,Sunite sheep and Prairie Tibetan sheep)sheep breeds were selected and sequenom SNP assay was applied to genotype two loci of DIO 2 gene.Then the association was analyzed between DIO 2 and litter size in Small Tail Han sheep.[Results]The results of qPCR showed that DIO 2 gene expressed in systemic tissues in sheep,but the expression level of this gene was higher in hypothalamus,pituitary,ovary,uterus and oviduct of Prairie Tibetan sheep than that in Small Tail Han sheep(P<0.05).From the result of genotyping,both genotype frequency and allele frequency were extremely different between year-round estrous and seasonal estrous sheep(P<0.01)at g.88484603G>C locus in DIO 2 gene.Population genetic analysis showed that the g.88484594T>C locus had moderate polymorphism(0.25<PIC<0.5)in Tan sheep and Celle Black sheep,and was at low polymorphism(PIC<0.25)in other sheep breeds,g.88484603G>C locus was at moderate polymorphism(0.25<PIC<0.5)in six populations of sheep.Theχ2 test indicated that the g.88484603G>C locus was under Hardy-Weinberg equilibrium(P>0.05)in six populations of sheep,but g.88484594T>C locus was not under Hardy-Weinberg equilibrium(P<0.05)in those sheep.Bioinformatics analysis showed that the mRNA secondary structure of g.88484603G>C locus had changed before and after mutation,and the minimum structural free energy was reduced,the structural stability was enhanced,and the secondary structure of protein had not changed,but there were some changes in the tertiary structure of protein.Association analysis indicated that the polymorphism of two SNP had no correlation with the litter size in Small Tail Han sheep(P>0.05).[Conclusions]DIO 2 gene is closely related to the seasonal reproduction of sheep,and the g.88484603G>C locus might provide a basis for seasonal breeding in sheep.