奶牛CRY1基因真核表达载体构建、生物信息学分析及组织表达谱研究

doi:10.16431/j.cnki.1671-7236.2023.03.003

摘要/Abstract

摘要： 【目的】扩增奶牛隐花色素昼夜节律调节因子1(cryptochrome circadian regulator 1,CRY1)基因全长编码区(coding sequence,CDS),构建该基因的真核表达载体并对其进行生物信息学分析,检测奶牛CRY1基因的表达谱,为后续探究CRY1蛋白的生物学功能提供参考。【方法】以奶牛肝脏组织cDNA为模板,PCR扩增CRY1基因CDS区,将其与酶切线性化的pcDNA3.1-3HA空质粒以同源重组法连接,重组质粒命名为pcDNA3.1-3HA-cCRY1;利用在线软件对奶牛CRY1基因进行生物信息学分析。将空质粒pcDNA3.1-3HA和重组质粒pcDNA3.1-3HA-cCRY1转染至HEK293T细胞,利用Western blotting技术检测奶牛CRY1基因在蛋白水平的表达;利用实时荧光定量PCR检测CRY1基因在奶牛不同组织中的表达量。【结果】试验成功获得1764 bp的奶牛CRY1基因CDS区序列,且成功构建pcDNA3.1-3HA-cCRY1真核表达载体。奶牛CRY1基因与牦牛、山羊、绵羊相似性在98%以上,且遗传距离较近。生物信息学分析表明,CRY1蛋白为碱性、亲水性蛋白,无跨膜结构与信号肽,存在45个潜在磷酸化位点,与CLOCK、ARNTL、FBXL3等多个蛋白存在互作。Western blotting结果表明,pcDNA3.1-3HA-cCRY1转染组在72 ku处出现明显的条带,而pcDNA3.1-3HA转染组在相应位置处无条带。实时荧光定量PCR结果表明,CRY1基因在奶牛心脏、肝脏、脾脏、肺脏等10个组织中均有表达,其中在心脏中的表达量最高,在颈斜方肌中的表达量最低。【结论】本研究成功构建了CRY1基因的真核表达载体,且在HEK293T细胞中实现了CRY1蛋白的过表达。CRY1蛋白是一种不稳定的胞内蛋白,可与多种蛋白互作,并参与昼夜节律调控、细胞代谢、糖异生等过程。CRY1基因在牛、羊等反刍动物间较为保守,在奶牛多种组织中均有表达。研究结果为深入探究CRY1蛋白在奶牛生物钟系统中的转录调控机制提供参考依据。

关键词: 奶牛; CRY1基因; 生物信息学; 组织表达谱

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

中图分类号:

李丹, 张海森, 王逸群, 高登科, 赵泓淙, 李超, 靳亚平, 陈华涛. 奶牛CRY1基因真核表达载体构建、生物信息学分析及组织表达谱研究[J]. 中国畜牧兽医, 2023, 50(3): 870-881.

LI Dan, ZHANG Haisen, WANG Yiqun, GAO Dengke, ZHAO Hongcong, LI Chao, JIN Yaping, CHEN Huatao. Eukaryotic Expression Vector Construction,Bioinformatics and Tissue Expression Profiles Analysis of CRY1 Gene in Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 870-881.

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