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

Abstract

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

CLC Number:

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.

References

[1] PATKE A,YOUNG M W,AXELROD S.Molecular mechanisms and physiological importance of circadian rhythms[J].Nature Reviews:Molecular Cell Biology,2020,21(2):67-84.
[2] DUNLAP J C.Molecular bases for circadian clocks[J].Cell,1999,96(2):271-290.
[3] CHEN H,GAO L,XIONG Y,et al.Circadian clock and steroidogenic-related gene expression profiles in mouse Leydig cells following dexamethasone stimulation[J]. Biochemical and Biophysical Research Communications,2017,483(1):294-300.
[4] CHAIX A,LIN T,LE H D,et al.Time-restricted feeding prevents obesity and metabolic syndrome in mice lacking a circadian clock[J].Cell Metabolism,2019,29(2):303-319.
[5] PAN X,MOTA S,ZHANG B.Circadian clock regulation on lipid metabolism and metabolic diseases[J].Advances in Experimental Medicine and Biology,2020,1276:53-66.
[6] HONMA S.Development of the mammalian circadian clock[J].European Journal of Neuroscience,2020,51(1):182-193.
[7] COX K H,TAKAHASHI J S.Circadian clock genes and the transcriptional architecture of the clock mechanism[J].Journal of Molecular Endocrinology,2019,63(4):R93-R102.
[8] 李冲.Cry1敲除对小鼠睾丸生殖功能的影响[D].重庆:重庆医科大学,2018. LI C.Cry1 deficiency damages mice testicular function[D].Chongqing:Chongqing Medical University,2018.(in Chinese)
[9] LAMIA K A,PAPP S J,YU R T,et al.Cryptochromes mediate rhythmic repression of the glucocorticoid receptor[J].Nature,2011,480(7378):552-556.
[10] ZHANG E E,LIU Y,DENTIN R,et al.Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis[J].Nature Medicine,2010,16(10):1152-1156.
[11] ZHOU J,TANG Z Y,SUN X L.RNF38 inhibits osteosarcoma cell proliferation by binding to CRY1[J].Biochemistry and Cell Biology,2021,99(5):629-635.
[12] 赵善江,郝海生,刘云祥,等.疾病对奶牛繁殖性能的影响及其研究进展Ⅲ:营养代谢性疾病[J].中国畜牧兽医,2020,47(11):3618-3625. ZHAO S J,HE H S,LIU Y X,et al.Effects of disease on reproductive performance in dairy cattle Ⅲ:Nutritional metabolic diseases[J].China Animal Husbandry & Veterinary Medicine,2020,47(11):3618-3625.(in Chinese)
[13] CAL-KAYITMAZBATIR S,KULKOYLUOGLU-COTUL E,GROWE J,et al.CRY1-CBS binding regulates circadian clock function and metabolism[J].FEBS Journal,2021,288(2):614-639.
[14] TOLEDO M,BATISTA-GONZALEZ A,MERHEB E,et al.Autophagy regulates the liver clock and glucose metabolism by degrading CRY1[J].Cell Metabolism,2018,28(2):268-281.
[15] JANG H,LEE G,KIM J B.SREBP1c-CRY1 axis suppresses hepatic gluconeogenesis upon insulin[J].Cell Cycle,2017,16(2):139-140.
[16] GRIEBEL G,RAVINET-TRILLOU C,BEESKE S,et al.Mice deficient in cryptochrome 1(Cry1^-/-) exhibit resistance to obesity induced by a high-fat diet[J].Frontiers in Endocrinology,2014,5:49.
[17] JANG H,LEE G Y,SELBY C P,et al.SREBP1c-CRY1 signalling represses hepatic glucose production by promoting FOXO1 degradation during refeeding[J].Nature Communications,2016,7:12180.
[18] 赵泓淙,高登科,江海圳,等.山羊生物钟基因CLOCK真核表达载体的构建和生物信息学分析[J].中国畜牧兽医,2021,48(12):4327-4338. ZHAO H C,GAO D K,JIANG H Z,et al.Construction of eukaryotic expression vector and bioinformatics analysis of circadian CLOCK gene in goats[J].China Animal Husbandry & Veterinary Medicine,2021,48(12):4327-4338.(in Chinese)
[19] 王逸群,高登科,赵泓淙,等.山羊NR1D1基因真核表达载体的构建及生物信息学分析[J].中国畜牧杂志,2022,58(10):215-222. WANG Y Q,GAO D K,ZHAO H C,et al.Construction of a eukaryotic expression vector in goat NR1D1 gene and its bioinformatics analysis[J].Chinese Journal of Animal Science,2022,58(10):215-222.(in Chinese)
[20] 高登科,赵泓淙,董浩,等.山羊RORα基因的克隆、表达载体构建及功能分析[J].畜牧兽医学报,2022,53(6):1779-1794. GAO D K,ZHAO H C,DONG H,et al.The cloning,expression vector construction and function analysis of goat RORα gene[J].Acta Veterinaria et Zootechnica Sinica,2022,53(6):1779-1794.(in Chinese)
[21] XIAO Y,ZHAO L,LI W,et al.Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells[J].Journal of Cellular Physiology,2021,236(9):6706-6725.
[22] HUMPHREY S J,JAMES D E,MANN M.Protein phosphorylation:A major switch mechanism for metabolic regulation[J].Trends in Endocrinology and Metabolism,2015,26(12):676-687.
[23] VAUGHAN M,JORDAN S D,DUGLAN D,et al.Phosphorylation of CRY1 serine 71 alters voluntary activity but not circadian rhythms in vivo[J].Journal of Biological Rhythms,2019,34(4):401-409.
[24] SIEPKA S M,YOO S H,PARK J,et al.Circadian mutant overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression[J].Cell,2007,129(5):1011-1023.
[25] BUSINO L,BASSERMANN F,MAIOLICA A,et al.SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins[J].Science,2007,316(5826):900-904.
[26] GODINHO S I,MAYWOOD E S,SHAW L,et al.The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period[J].Science,2007,316(5826):897-900.
[27] WILKINSON G W,AKRIGG A.Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector[J].Nucleic Acids Research,1992,20(9):2233-2239.
[28] 赵明旺.BMAL1在牦牛颗粒细胞中的表达及功能研究[D].青海:青海大学,2020. ZHAO M W.Study on expression and function of BMAL1 in granulosa cells of yak[D].Qinghai:Qinghai University,2020.(in Chinese)
[29] 高磊,甘尚权,杨井泉,等.绵羊发情周期不同组织Cry1 mRNA转录水平相对定量研究[J].遗传,2013,35(1):85-92. GAO L,GAN S Q,YANG J Q,et al.Relative quantification of mRNA transcription of Cry1 in different tissue of sheep in oestrous cycle by real-time quantitative PCR[J].Hereditas,2013,35(1):85-92.(in Chinese)
[30] 习瓒娜.瘤胃上皮挥发性脂肪酸转运蛋白的节律性及PER2对其调控的研究[D].扬州:扬州大学,2021. XI Z N.Rhythm of volatile fatty acid transporters in rumen epithelium and its regulation by period2[D].Yangzhou:Yangzhou University,2021.(in Chinese)
[31] 陈健楠.牦牛CRY1基因功能研究[D].兰州:甘肃农业大学,2019. CHEN J N.Study on the function of CRY1 gene in yak[D].Lanzhou:Gansu Agricultural University,2019.(in Chinese)

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

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