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

doi:10.11843/j.issn.0366-6964.2023.01.013

Abstract

Abstract: 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-cNR1D1was 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.

Key words: ruminants, circadian clock, nuclear receptor, reproduction, expression profiles

CLC Number:

S823.91

WANG Yiqun, LIU Zupei, LI Yating, ZHANG Haisen, LI Dan, JIN Yaping, CHEN Huatao. The Dairy Cow NR1D1 Gene’s Eukaryotic Expression Vector Construction, Expression Profile and Its Ovarian Localization[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 133-145.

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

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