奶牛NR1D1基因的真核表达载体构建、表达谱及其在卵巢组织的定位

The Dairy Cow NR1D1 Gene’s Eukaryotic Expression Vector Construction,Expression Profile and Its Ovarian Localization

旨在克隆奶牛核受体亚家族1 D组成员1(nuclear receptor subfamily 1 group D member 1,NR1D1)基因的蛋白编码序列(coding sequence, CDS),构建其真核表达载体,预测分析NR1D1基因的生物学功能,并检测该基因的组织表达谱及其在卵巢组织的表达分布。本研究以5头健康的24月龄雌性奶牛为试验动物,PCR扩增带有同源臂的奶牛NR1D1基因CDS区全长片段,利用同源重组法将目的片段连接至线性化的pcDNA3.1-Puro-N-3HA空载体,构建重组质粒,利用酶切、PCR及测序对重组质粒进行鉴定。结合质粒测序结果,利用生物信息学软件预测分析该基因的生物学功能。将鉴定正确的质粒转染至HEK293T细胞,利用实时荧光定量PCR(qPCR)与Western blotting技术检测NR1D1基因在mRNA与蛋白水平的表达。此外,利用qPCR检测奶牛NR1D1基因的组织表达谱,并利用免疫组织化学技术检测NR1D1蛋白在奶牛卵巢组织的表达分布。结果,成功获得带有同源臂的奶牛NR1D1基因的CDS区全长片段(1 884 bp),酶切、PCR及测序结果表明pcDNA3.1-3HA-cNR1D1真核表达载体构建成功。生物信息学分析结果显示,NR1D1蛋白不存在跨膜结构,为核蛋白,其核定位序列存在于第200位氨基酸附近,且NR1D1蛋白中存在86个潜在的磷酸化位点;奶牛与小鼠NR1D1蛋白三级结构较为相似,奶牛NR1D1蛋白可能在奶牛生殖、代谢与免疫等生理过程中发挥重要作用。将pcDNA3.1-3HA-cNR1D1转染至HEK293T细胞,奶牛NR1D1基因在mRNA与蛋白水平均成功过表达。qPCR结果显示,NR1D1基因在奶牛瘤胃、结肠与肝中表达量显著高于其他组织;免疫组织化学结果显示,奶牛NR1D1表达于不同发育时期卵泡的颗粒细胞中。本研究成功构建了奶牛NR1D1基因真核表达载体,且在HEK293T细胞成功实现了该基因的过表达,对奶牛NR1D1基因及其编码蛋白的理化性质与生物学特性进行了预测分析,并获得了该基因的组织表达谱及在奶牛卵巢组织的表达分布情况,为深入探究奶牛NR1D1基因的生物学功能提供了前期基础与关键材料。

This study aimed to clone the coding sequence(CDS) of the nuclear receptor subfamily 1 group D member 1 gene(NR1D1) in dairy cows, construct its eukaryotic expression vector, predict and analyze the biological function of the NR1D1 gene, detect the tissue expression profiles of the gene and its expression distribution in ovary. In this study, 5 healthy female dairy cows(24 months old) were used as experimental animals. The full-length fragment of the CDS region in dairy cow NR1D1 gene with homologous arms was amplified by PCR, and the target fragment was ligated to the linearized pcDNA3.1-Puro-N-3HA vector by homologous recombination to construct a recombinant plasmid. Recombinant plasmids were identified by enzyme digestion, PCR and sequencing techniques. Combined with the results of plasmid sequencing, bioinformatics softwares were used to predict and analyze the biological function of NR1D1. The correctly identified plasmid was transfected into HEK293T cells, and the expression of NR1D1 gene at mRNA and protein levels was detected by real-time quantitative PCR(qPCR) and Western blotting. In addition, qPCR was used to detect the expression profile of dairy cow NR1D1 gene in different tissues, and immunohistochemistry was used to focus on its expression distribution in ovary. The full-length fragment of the CDS(1 884 bp) region of NR1D1 gene in dairy cow with homology arms was obtained by PCR amplification. The results of restriction enzyme digestion, PCR identification and sequencing showed that the eukaryotic expression vector pcDNA3.1-3HA-cNR1D1 was successfully constructed. The results of bioinformatics analysis showed that the NR1D1 protein had no transmembrane structure and was a nucleoprotein, its nuclear localization sequence existed near the 200th amino acid, and there were 86 potential phosphorylation sites in NR1D1 protein. The tertiary structure of cow and mouse NR1D1 protein was similar and the NR1D1 protein may play an important role in the physiological process of reproduction, metabolism and immunity in dairy cow. The pcDNA3.1-3HA-cNR1D1 was transfected into HEK293T cells, and the cow NR1D1 gene was successfully overexpressed at both mRNA and protein levels. The qPCR results showed that the expression of NR1D1 gene in dairy cow’s rumen, colon and liver was significantly higher than other tissues. Immunohistochemical results showed that NR1D1 was mainly expressed in granulosa cells of follicles in different developmental stages. This study successfully constructed dairy cow NR1D1 gene’s eukaryotic expression vector, and successfully overexpressed NR1D1 in HEK293T cells. The tissue expression profile of NR1D1 and its expression distribution in the ovary of dairy cows were preliminarily predicted and analyzed. This study provided the preliminary basis and key materials for in-depth exploration of NR1D1’s biological function.