miR-23a/27a/24基因簇g.65307469G>A突变对大白猪产仔数和启动子活性的影响

The effect of g.65307469G>A variant in miR-23a/27a/24 cluster on litter size traits in Large White sow and promoter activity

[目的]本文旨在研究大白猪miR-23a/27a/24基因簇启动子突变位点多态性与产仔数性状的关系及潜在机制,为母猪繁殖性状的分子育种提供新的潜在遗传标记。[方法]利用混池测序技术筛选猪miR-23a/27a/24基因簇启动子突变位点,直接测序法对大白猪群体(n=345)miR-23a/27a/24基因簇突变位点进行基因分型,计算遗传多样性;用线性模型对突变位点多态性与产仔数性状进行关联性分析;构建不同等位基因类型启动子载体,转染猪卵巢颗粒细胞,通过检测荧光素酶活性分析突变对启动子活性的影响;用生物信息学方法预测不同等位基因类型启动子潜在结合的差异转录因子,用荧光素酶活性分析差异转录因子对不同等位基因类型启动子活性的影响。[结果]在猪miR-23a/27a/24基因簇启动子-648 nt(Chr.2,65307469 nt)处发现1个新的G/A突变,命名为g.65307469G>A。在大白猪群体中发现3种基因型(GG、GA和AA),其中GG为优势基因型(89.57%),G等位基因为优势等位基因(94.64%)。关联性分析发现,GA基因型母猪的总产仔数(TNB)比GG基因型母猪每胎高0.56头(P<0.05)。荧光素酶活性分析结果显示G等位基因类型启动子活性显著高于A等位基因类型(P<0.05)。在不同等位基因类型启动子间发现1个潜在结合的差异转录因子死亡相关蛋白1(THAP1),成功构建猪THAP1基因过表达载体,共转试验结果显示转录因子THAP1对不同等位基因类型启动子活性均无显著影响(P>0.05)。[结论]miR-23a、miR-27a和miR-24是大白猪产仔数性状的候选基因,g.65307469G>A突变抑制miR-23a/27a/24基因簇的启动子活性,但与转录因子THAP1无关。

[Objectives]The paper aimed to study the relationship between the polymorphisms of variants in miR-23a/27a/24 gene cluster promoter and litter size traits and its potential mechanism in Large White sow,and to provide a new potential genetic marker for molecular breeding of reproductive traits in sow.[Methods]The pooled sequencing was used to screen for variants in the promoter region of porcine miR-23a/27a/24 gene cluster,and the direct sequencing was used to detect genotype variants in a Large White sows population(n=345).The association between the polymorphisms and litter size traits was analyzed by a linear model.Different allelic promoter vectors were constructed and transfected into porcine ovarian granulosa cells(GC),and the effect of the variants on promoter activity was analyzed by luciferase activity assay.Bioinformatic methods were used to predict the potential binding of differential transcription factors in different allelic promoter,and the effect of differential transcription factors on the activity of different allelic promoters was verified by luciferase activity assay.[Results]A novel G/A variant,named g.65307469G>A,was identified at-648 nt(Chr.2,65307469 nt)in the promoter region of porcine miR-23a/27a/24 gene cluster.Three genotypes(GG,GA and AA)were observed in the Large White sow population,of which GG was the dominant genotype(89.57%)and the G allele was the dominant allele(94.64%).The association analysis revealed that the total number of piglets born(TNB)of sows with GA genotype was 0.56 higher than that of sows with GG genotype per litter(P<0.05).Luciferase activity assay showed that the activity of promoter region with G allele was significantly higher than that of promoter region with the A allele(P<0.05).A potentially binding site for transcription factor thanatos-associated protein 1(THAP1)was found in the promoter regions with the G allele,but not A allele,and a porcine THAP1 overexpression vector was successfully constructed.The results of the co-transformation experiment revealed that the THAP1 had no significant effect on the activity of the promoter regions with the G or A alleles(P>0.05).[Conclusions]The miR-23a/27a/24 gene cluster was a candidate gene cluster for litter size traits in Large White sow.The g.65307469G>A mutation affected the promoter activity of miR-23a/27a/24 gene cluster,but was not related to the transcription factor THAP1.