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Three Complete Linkage SNPs of GDF9 Gene Affect the Litter Size Probably Mediated by OCT1 in Hu Sheep

文献类型： 外文期刊

作者： Li, Yinxia 1 ; Jin, Wenwen 4 ; Wang, Yue 1 ; Zhang, Jun 1 ; Meng, Chunhua 1 ; Wang, Huili 1 ; Qian, Yong 1 ; Li, Qifa 4 ; Ca 1 ;

作者机构： 1.Jiangsu Acad Agr Sci, Inst Anim Sci, Nanjing 210014, Peoples R China

2.Jiangsu Prov Platform Conservat & Utilizat Agr Ge, Nanjing, Peoples R China

3.Minist Agr, Key Lab Crop & Anim Integrated Farming, Nanjing, Peoples R China

4.Nanjing Agr Univ, Coll Anim Sci & Technol, Nanjing 210095, Peoples R China

关键词： GDF9; promoter region; OCT1; single-nucleotide polymorphism; apoptosis

期刊名称：DNA AND CELL BIOLOGY （ 影响因子：3.311； 五年影响因子：3.557 ）

ISSN： 1044-5498

年卷期： 2020 年 39 卷 4 期

页码：

收录情况： SCI

摘要： Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).

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