Jiangsu Academy of Agricultural Sciences, Institute of Animal Science, Nanjing, China.
The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing, China.
DNA Cell Biol. 2020 Apr;39(4):563-571. doi: 10.1089/dna.2019.4984. Epub 2020 Feb 19.
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 gene. In this study, the SNPs in the promoter region were explored and their transcription mechanisms in regulating expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. 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 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 (), 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 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).
生长分化因子（GDF）9 基因参与调控动物的生殖性状，但关于该基因的启动子、单核苷酸多态性（SNP）、转录因子结合位点及其调控机制知之甚少。本研究旨在探讨其启动子区的 SNP，并分析其在调控 GDF9 表达中的转录机制。采集 267 只湖羊耳部组织，提取基因组 DNA。采用 PCR 扩增 GDF9 启动子区，测序鉴定 SNP 基因型。采用 SPSS16.0 软件分析基因型与产羔数的关联性。采用流式细胞术检测细胞凋亡，双荧光素酶报告基因检测启动子活性。通过 PCR 扩增获得湖羊 GDF9 基因 1789bp 启动子区，荧光素酶活性检测显示，-725 到-309bp 区域存在负调控元件，-309 到+43bp 区域存在正调控元件。检测到-534A/G、-407T/G 和-332C/T 三个完全连锁 SNP，形成 3 种基因型（AA、AB 和 BB）。关联分析表明，AA 基因型母羊的平均胎产羔数大于 BB 基因型。-534A/G 突变创造了一个新的八聚体转录因子 1（OCT1）结合位点，Annexin V FITC/PI 流式细胞术检测显示 OCT1 促进绵羊卵巢颗粒细胞凋亡。OCT1 过表达显著抑制两种基因型的荧光素酶活性，且 pGL3-BB 的抑制作用高于 pGL3-AA。GDF9 基因的 3 个完全连锁 SNP 可能通过与 OCT1 在-534GG 基因型上的结合来抑制启动子活性，形成 OCT1 与 CCAAT/增强子结合蛋白（C/EBP）的复合物，从而调节湖羊的产羔数。
