绵羊GDF9基因mRNA、DNA和调控区序列克隆及其在11个品种中遗传多态性检测

Cloning and Genetic Polymorphism Analysis of mRNA,DNA and Regulatory Region of Ovine GDF9 Gene in 11 Breeds

本研究旨在克隆绵羊生长分化因子9（Growth differentiation factor 9,GDF9）基因mRNA、DNA和调控区全序列,并检测其在11个绵羊品种中的遗传多态性,探究GDF9基因与绵羊多羔性状的关系。首先应用RACE和PCR技术克隆GDF9基因全长序列,其次利用SNaPshot分型技术检测11个绵羊品种GDF9基因遗传多态性。结果显示,克隆获得GDF9基因mRNA全长1 852bp（GenBank序列号：KR063137）,编码区两侧翼分别具有5′-UTR58bp（1~58nt）和3′-UTR 432bp（1 421~1 852nt）。进一步克隆获得长为2 898bp的DNA序列和2 304bp的调控区序列,分析发现调控区序列比数据库序列（NC_019462.1）多两个长片段。序列比对发现了已知突变G260A（G1）和调控区新突变-2078C〉G。分型结果显示,11个绵羊品种均不含FecGE、FecGH、FecGT、FecGF、FecGV突变,而G260A和-2078C〉G广泛存在于除草原型藏羊外的各绵羊品种中,G260A表现3种基因型（AA、BB和AB）,首次在小尾寒羊和山谷型藏羊中检测到BB型突变纯合子。-2078C〉G也存在3种基因型,基因型结果表明该位点与G260A完全连锁。关联分析结果显示,小尾寒羊AB型产羔数显著高于AA型（P〈0.05）。本研究完善了绵羊GDF9mRNA、DNA和调控区全长序列,为进一步研究GDF9基因功能奠定了基础。同时在不同绵羊品种中发现了G260A和-2078C〉G突变,其中G260A作为潜在的有效遗传标记可用于提高绵羊的产羔数。

The aim of this study was to clone the mRNA,DNA and regulatory region sequence of ovine GDF9 gene,detect its genetic polymorphism in 11 sheep breeds,and search for the molecular genetic markers related to sheep litter size.The RACE and PCR technologies were used to clone full-length sequence of ovine GDF9 gene,and SNaPshot was performed to detect the polymorphisms of GDF9 gene in 11 breeds.A 1 852 bp full-length mRNA of ovine GDF9gene（GenBank No.：KR063137）was obtained,which contains 58 bp 5′-UTR and 432 bp 3′-UTR.In addition,2 898 bp of DNA sequence and 2 304 bp of regulatory region sequence were amplified.Compared with the sequence of NC_019462.1,the result had 2more fragments in regulatory region.The genetic polymorphism analysis showed that a known mutation G260A（G1）and a novel mutation-2078〉CG within regulatory region were identified.The genotyping results showed that11 sheep breeds were free of FecGE,FecGH,FecGT,FecGF,FecGV mutations,while the G260 A and-2078〉CG were widespread in all sheep except Prairie Tibetan sheep.G260 Acontained 3genotypes AA,BB and AB,but the BB genotype only presented in Small-tail Han sheep and Valley Tibetan sheep.-2078〉CG also included 3genotypes,in which the genotype and allele frequencies were exactly the same with G260 A.It seems that-2078〉CG was complete linkage with G260 A.Association analysis in Small-tail Han sheep showed that the litter size of individuals with AB genotype was significantly higher than that of AA genotype（P〈0.05）.We improved the mRNA,DNA and regulatory region sequences of ovine GDF9,which provided a foundation for further functional study of GDF9 gene.Meanwhile,G260 A and-2078〉CG mutations were found in different sheep breeds,and G260 Asite could be a potential genetic marker for improving litter size in sheep.