FSHβ基因5′调控区多态性与小尾寒羊产羔数的关系

Polymorphism of 5′ regulatory region of FSHβ gene and its relationship with litter size of Small Tail Han sheep

利用反转脉冲电泳改进的单链构象多态(PCR-SSCP)方法对促卵泡素β(FSHβ)亚基基因5′调控区近端1.5kb的序列进行多态性检测,分析该基因对小尾寒羊产羔数的影响。结果表明:引物P3、P5和P6扩增片段均存在多态性,在小尾寒羊中均检测到3种基因型。测序结果显示引物P3的扩增片段存在C、D2种等位基因;相对于野生型,C、D等位基因均在-899bp处发生了1个TCG→GTC的三碱基突变;此外,C等位基因在-933bp处发生了1个T→A的单碱基突变。引物P5的扩增片段存在A、B2种等位基因,B等位基因序列与GenBank中序列完全一致,定义为野生型;而A等位基因则发生了A-438G、T-411C和T-375G的3个单碱基突变;还发现A等位基因在-402bp处存在1个单碱基的缺失(T)。引物P6的扩增片段存在E、F2种等位基因,F等位基因与GenBank中序列一致,E等位基因在-181bp处发生了1个T→C的单碱基突变。统计发现小尾寒羊A、C、E等位基因频率明显高于B、D、F,基因型频率AA>AB>BB、CC>CD>DD、EE>EF>FF;且引物P3遗传变异最大,P5遗传变异最小;小尾寒羊产羔数的最小二乘均值关系为AA>AB>BB、CC>CD>DD、EE>EF>FF,但3种基因型间产羔数差异均不显著(P>0.05)。

The FSH gene was studied as a candidate gene for prolificacy in Small Tail Han sheep. Single nucleotide polymorphisms （SNPs） of about 1.5 kb sequence of the proximal 5＇regulatory region of follicle-stimulating hormone FSH geneβ subunit were detected by polymerase chain reaction-single strand conformation polymorphism. The result indicated that the product amplified by primer P3, P5 and P6 displayed polymorphism. Three genotypes were detected in each primer. Sequencing revealed that there were two alleles （C and D） of product amplified by primer P3. Compared with wild type, there was one three-base mutation （TCG→GTC） at -899 bp of FSHβ gene in the D allele. Besides this muta- tion, a single nucleotide mutation （T→A） at-933 bp in the C allele was also detected. Two couples of alleles （A and B, E and F） showed in product amplified by primer P5 and primer P6. Sequences of B allele and F allele were completely consistent with GenBank, which were defined as wild type; while there were three single-base mutations （A- 438G, T-411C and T-375G） in the A allele, and one single-base mutations （T-181C） in the E allele. In addition, a single base deletion （T） at -402 bp was also found in the A allele. Statistic results revealed that A, C, E allele frequencies were significantly higher than the B, D, F allele frequency. AA, CC, EE genotype frequencies were higher than AB,CD, EF genotype frequencies, and BB, DD, FF genotype frequencies were the lowest ones in Small Tail Han sheep. Statistics results showed that it varied most in primer P3 and varied least in primer P5. The least squares value of litter size in Small Tail Han sheep were AA〉AB〉BB, CC〉CD〉 DD, EE〉EF〉FF, but differences of LSM value of litter size between those genotypes were not significant （P〈0.05）.