China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (3): 870-881.doi: 10.16431/j.cnki.1671-7236.2023.03.003

• Biotechnology • Previous Articles     Next Articles

Eukaryotic Expression Vector Construction,Bioinformatics and Tissue Expression Profiles Analysis of CRY1 Gene in Dairy Cows

LI Dan1, ZHANG Haisen1,2, WANG Yiqun1, GAO Dengke1,2, ZHAO Hongcong1,2, LI Chao1,2, JIN Yaping1,2, CHEN Huatao1,2   

  1. 1. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
    2. Key Laboratory of Animal Biotechnology of the Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling 712100, China
  • Revised:2022-10-27 Online:2023-03-05 Published:2023-03-02

Abstract: 【Objective】 The purpose of this study was to amplify the full-length coding sequence (CDS) of cryptochrome circadian regulator 1 (CRY1) gene in dairy cows,construct its eukaryotic expression vector and conduct bioinformatics analysis,and detect the expression profiles of CRY1 gene in dairy cows,so as to provide a reference for subsequent exploration of the biological function of CRY1 protein.【Method】 The CDS region of CRY1 gene was amplified by PCR using the cDNA template from liver in dairy cows.The CDS of CRY1 gene was connected to the enzyme digesting linearized pcDNA3.1-3HA empty plasmid by homologous recombination method.The recombinant plasmid with correct sequence was named pcDNA3.1-3HA-cCRY1.Bioinformatics software was used to analyze CRY1 gene in dairy cows.The pcDNA3.1-3HA empty plasmid and pcDNA3.1-3HA-cCRY1 recombinant plasmid were transfected into HEK293T cells,respectively.Western blotting was used to detect the expression of CRY1 protein.Real-time quantitative PCR was used to detect the expression profile of CRY1 gene in different tissues of dairy cows.【Result】 The CDS region (1 764 bp) of CRY1 gene was successfully obtained,and the eukaryotic expression vector pcDNA3.1-3HA-cCRY1 was successfully constructed.The CDS region of CRY1 gene in dairy cows was more than 98% similarity to Bos mutus,Capra hircus and Ovis aries,and their genetic distance were close relatively.Bioinformatics analysis showed that CRY1 protein was alkaline and hydrophilic protein,without transmembrane domain and signal peptide.CRY1 protein had 45 potential phosphorylation sites,and interacted with CLOCK,ARNTL,FBXL3 and other proteins.Western blotting result showed that the pcDNA 3.1-3HA-cCRY1 transfection group had a distinct band at 72 ku,while the pcDNA 3.1-3HA transfection group had no band at the corresponding locations.Real-time quantitative PCR results showed that CRY1 gene in dairy cows was expressed in 10 tissues such as heart,liver,spleen and lung,with the highest expression in heart and the lowest expression in trapezius cervical muscle.【Conclusion】 In this study,the eukaryotic expression vector of CRY1 gene was successfully constructed,and the overexpression of CRY1 protein was achieved in HEK293T cells.CRY1 protein was an unstable intracellular protein that could interact with multiple proteins and participate in circadian rhythm regulation,cell metabolism,gluconeogenesis and other processes. CRY1 gene was conserved among ruminants such as Bos taurus and Ovis aries,which was distributed in various tissues of dairy cows.The results provided a reference for further exploration of the transcriptional regulation mechanism of CRY1 protein in the biological clock system of dairy cows.

Key words: dairy cows; CRY1 gene; bioinformatics; tissue expression profiles

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