山羊C/EBPα、C/EBPβ和C/EBPδ基因克隆及表达模式研究

doi:10.7668/hbnxb.20190869

摘要/Abstract

摘要： 在克隆获得山羊C/EBPα、C/EBPβ和C/EBPδ基因序列基础上分析该家族成员序列特征，明确组织表达特性，并揭示3个基因在山羊肌内前体脂肪细胞分化过程中的表达情况。采用RT-PCR方法克隆山羊C/EBPα、C/EBPβ和C/EBPδ基因序列，利用相关的生物软件进行生物信息学分析，利用qPCR检测3个基因在山羊各组织及山羊肌内前体脂肪细胞成脂分化中的表达情况。克隆得到C/EBPα、C/EBPβ和C/EBPδ基因序列分别为1 062，1 056，873 bp，所编码的蛋白均为不稳定亲水碱性蛋白，均无信号肽和跨膜结构域。C/EBPα在皮下脂肪中表达水平最高，C/EBPβ和C/EBPδ均在肺中表达水平最高。在前体脂肪细胞分化过程中C/EBPα和C/EBPδ均在前体脂肪细胞分化前表达水平最高，随着分化时间的增加表达水平降低，C/EBPβ在肌内前体脂肪细胞中的表达随着诱导分化天数的增加而增加，在分化7 d时表达水平最高。基于以上研究，推测3个基因在山羊肌内脂肪细胞分化过程中发挥着重要作用。

关键词: 山羊, 基因克隆, 表达, 细胞分化

Abstract: This experiment was conducted to study the sequence characteristics, physicochemical properties of goat C/EBPα, C/EBPβ and C/EBPδ genes and proteins, and the expression patterns in goat tissues and in the differentiation of goat intramuscular precursor adipocytes.The C/EBPα, C/EBPβ and C/EBPδ of goat were cloned by RT-PCR method and the biological characteristics were analyzed using the relevant biological software. qPCR was used to detect the expression levels of C/EBPα, C/EBPβ and C/EBPδ mRNA in different tissues and preadipocyte of goat. The results showed that the sequence of C/EBPα was 1 062 bp, C/EBPβ was 1 056 bp and C/EBPδ was 873 bp. The proteins encoded by C/EBPα, C/EBPβ and C/EBPδ were all unstable hydrophilic basic proteins, and had no signal peptide and transmembrane domain. The expression of three genes was detected in different tissues of goats. The expression level of C/EBPα was highest in subcutaneous fat, and the expression pattern of C/EBPβ and C/EBPδ was similar, and the expression level was highest in lung.The expression patterns of C/EBPα and C/EBPδ were similar in the differentiation of adipose precursor cells, and the expression level was the highest at 0 d after differentiation, and the expression level was decreased with the increase of differentiation time. C/EBPβ was found in intramuscular precursor fat. The expression of C/EBPβ in the cells increased with the number of days of induced differentiation, and the expression level was highest at 7 days of differentiation. Based on the above studies, it is speculated that the three genes play an important role in the process of goat adipose differentiation.

Key words: Goat, Gene cloning, Expression, Adipose differentiation

中图分类号:

Q78
S826

池永东, 王永, 赵越, 朱江江, 林亚秋. 山羊C/EBPα、C/EBPβ和C/EBPδ基因克隆及表达模式研究[J]. 华北农学报, 2020, 35(5): 231-238. doi: 10.7668/hbnxb.20190869.

CHI Yongdong, WANG Yong, ZHAO Yue, ZHU Jiangjiang, LIN Yaqiu. Study on Cloning and Expression Patterns of Goat C/EBPα, C/EBPβ and C/EBPδ Genes[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(5): 231-238. doi: 10.7668/hbnxb.20190869.

http://www.hbnxb.net/CN/Y2020/V35/I5/231

参考文献

[1] 盘道兴, 王振, 杨茂林, 廖乔平, 杨昌平, 吴宇萍, 王金洲, 刘若余. 不同品种猪PPARγ 和C/EBPα 基因表达规律与肌内脂肪含量的相关[J]. 中国农业科学, 2017, 50(1):171-182. doi:10.3864/j.issn.0578-1752.2017.01.015.
Pan D X, Wang Z, Yang M L, Liao Q P, Yang C P, Wu Y P, Wang J Z, Liu R Y. Association of the PPARγ and C/EBPα gene expression with intramuscular fat content in different varieties of pig[J]. Scientia Agricultura Sinica,2017,50(1):171-182.
[2] Cirilli M, Bereshchenko O, Ermakova O, Nerlov C. Insights into specificity, redundancy and new cellular functions of C/EBPα and C/EBPb transcription factors through interactome network analysis[J]. Biochim et Biophysica Acta(BBA) General Subjects, 2017, 1861(2):467-476. doi:10.1016/j.bbagen.2016.10.002.
[3] Marchwicka A, Marcinkowska E. Regulation of expression of CEBP genes by variably expressed vitamin D receptor and retinoic acid receptor α in human acute myeloid leukemia cell lines[J]. International Journal of Molecular Sciences, 2018, 19(7):1907-1918. doi:10.3390/ijms19071918.
[4] Friedman A D. C/EBPα in normal and malignant myelopoiesis[J]. International Journal of Hematology, 2015, 101(4):330-341. doi:10.1007/s12185-015-1764-6.
[5] Tanaka S, Tanaka K, Magnusson F, Chung Y, Martinez G J, Wang Y H, Nurieva R I, Kurosaki T, Dong C. CCAAT/enhancer-binding protein α negatively regulates IFN-γ expression in T cells[J]. The Journal of Immunology, 2014, 193(12):6152-6160. doi:10.4049/jimmunol.1303422.
[6] Braissant O, Foufelle F, Scotto C, Dauça M, Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs):tissue distribution of PPAR-α,-β, and-γ in the adult rat[J]. Endocrinology,1996,137(1):354-366.doi:10.1210/endo.137.1.8536636.
[7] Ng C W S, Kosmo B, Lee P L, Lee C K, Guo J X, Chen Z J, Chiu L, Lee H K, Ho S, Zhou J B, Lin M X, Tan K M L, Ban K H K, Tan T W, Chng W J, Yan B. CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay[J]. Journal of Clinical Pathology, 2018, 71(6):522-531. doi:10.1136/jclinpath-2017-204825.
[8] Umek R M, Friedman A D, Mcknight S L. CCAAT-enhancer binding protein:a component of a differentiation switch[J]. Science, 1991, 251(4991):288-292. doi:10.1126/science.1987644.
[9] Feng R, Desbordes S C, Xie H F, Tillo E S, Pixley F, Stanley E R, Graf T. PU.1 and C/EBP α/β convert fibroblasts into macrophage-like cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(16):6057-6062. doi:10.1073/pnas.0711961105.
[10] Xie H F, Ye M, Feng R, Graf T. Stepwise reprogramming of B cells into macrophages[J]. Cell, 2004, 117(5):663-676. doi:10.1016/s0092-8674(04)00419-2.
[11] Rapino F, Robles E F, Richter-Larrea J A, Kallin E M, Martinez-Climent J A, Graf T. C/EBPα induces highly efficient macrophage transdifferentiation of B lymphoma and leukemia cell lines and impairs their tumorigenicity[J]. Cell Reports, 2013, 3(4):1153-1163. doi:10.1016/j.celrep.2013.03.003.
[12] Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family[J]. Embo Journal, 1990, 9(6):1897-1906. doi:10.1002/j.1460-2075.1990.tb08316.x.
[13] He Y F, Liu F Y, Zhang W X. Tangeritin inhibits adipogenesis by down-regulating C/EBPα, C/EBPβ, and PPARγ expression in 3T3-L1 fat cells[J]. Genetics & Molecular Research Gmr, 2015, 14(4):13642-13648. doi:10.4238/2015.October.28.26.
[14] Salma N, Xiao H, Imbalzano A N. Temporal recruitment of CCAAT/enhancer-binding proteins to early and late adipogenic promoters in vivo[J]. Journal of Molecular Endocrinology,2006, 36(1):139-151. doi:10.1677/jme.1.01918.
[15] Mori T, Sakaue H, Iguchi H, Gomi H, Okada Y, Takashima Y, Nakamura K, Nakamura T, Yamauchi T, Kubota N, Kadowaki T, Matsuki Y, Ogawa W, Hiramatsu R, Kasuga M. Role of Krüppel-like factor 15(KLF15) in transcriptional regulation of adipogenesis[J]. Journal of Biological Chemistry, 2000, 44(11):12867-12875. doi:10.1074/jbc.M410515200.
[16] Yeh W C, Cao Z, Classon M, McKnight S L. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins[J]. Genes & Development, 1995, 9(2):168-181. doi:10.1101/gad.9.2.168.
[17] 许晴,林森,朱江江,王永,林亚秋. 山羊肌内前体脂肪细胞诱导分化过程中内参基因的表达稳定性分析[J]. 畜牧兽医学报, 2018, 49(5):907-918. doi:10.11843/j.issn.0366-6964.2018.05.005.
Xu Q, Lin S, Zhu J J, Wang Y, Lin Y Q. The expression stability analysis of reference genes in the process of goat intramuscular preadipocytes differentiation in goat[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(5):907-918.
[18] Lee H, Kim H J, Lee Y J, Lee M Y, Choi H, Lee H, Kim J W. Krüppel-Like factor KLF8 plays a critical role in adipocyte differentiation[J]. PLoS One, 2012, 7(12):52474-52481. doi:10.1371/journal.pone.0052474.
[19] Zhang Q Y, Ramlee M K, Brunmeir R, Villanueva C J, Halperin D, Xu F. Dynamic and distinct histone modifications modulate the expression of key adipogenesis regulatory genes[J]. Cell Cycle, 2012, 11(23):4310-4322. doi:10.4161/cc.22224.
[20] Schroeder-Gloeckler J M, Rahman S M, Janssen R C, Qiao L P, Shao J H, Roper M, Fischer S J, Lowe E, Orlicky D J, McManaman J L, Palmer C, Gitomer W L, Huang W, O'Doherty R M, Becker T C, Klemm D J, Jensen D R, Pulawa L K, Eckel R H, Friedman J E.CCAAT/enhancer-binding protein β deletion reduces adiposity, hepatic steatosis, and diabetes in Lepr^db/db mice[J]. Journal of Biological Chemistry, 2007, 282(21):15717-15729. doi:10.1074/jbc.M701329200.
[21] 王万霞. 鸭C/EBPα, C/EBPβ和C/EBPδ基因的克隆及在脂肪组织中的发育性表达研究[D].成都:四川农业大学, 2012.
Wang W X. Cloning of duck C/EBPα, C/EBPβ, C/EBPδ genes and developmental expression in duck fat tissues[D]. Chengdu:Sichuan Agricultural University, 2012.
[22] 王娉. 鸡脂肪分化转录因子PPARγ和C/EBPα抗体制备及功能研究[D].乌鲁木齐:新疆大学, 2007.
Wang P. Ab preparation by chicken PPARγ and C/EBPα genes immunization and their function[D]. Urumqi:Xinjiang University, 2007.
[23] Moseti D, Regassa A, Kim W K. Molecular regulation of adipogenesis and potential anti-adipogenic bioactive molecules[J]. International Journal of Molecular Sciences, 2016, 17(1):124-147.doi:10.3390/ijms17010124.
[24] Lekstrom-Himes J, Xanthopoulos K G. Biological role of the CCAAT/enhancer-binding protein family of transcription factors[J]. Journal of Biological Chemistry, 1998, 273(44):28545-28548. doi:10.1074/jbc.273.44.28545.
[25] Poli V, Mancini F P, Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP[J]. Cell, 1990, 63(3):643-653. doi:10.1016/0092-8674(90)90459-r.
[26] Descombes P, Chojkier M, Lichtsteiner S, Falvey E, Schibler U. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein[J]. Genes Dev, 1990, 4(9):1541-1551. doi:10.1101/gad.4.9.1541.
[27] Kinoshita S, Akira S, Kishimoto T. A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6[J]. Proceedings of the National Academy of Sciences of the United States of America,1992,89(4):1473-1476. doi:10.1073/pnas.89.4.1473.
[28] Cao Z, Umek R M, Mcknight S L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells[J]. Genes Dev, 1991, 5(9):1538-1552. doi:10.1101/gad.5.9.1538.
[29] Rosen E D, Spiegelman B M. Molecular regulation of adipogenesis[J]. Annu Rev Cell Dev Biol, 2000, 16(4):145-171. doi:10.1146/annurev.cellbio.16.1.145.
[30] Liu Y, Peng W Q, Guo Y Y, Liu Y, Tang Q Q, Guo L. Krüppel-like factor 10(KLF10) is transactivated by the transcription factor C/EBPβ and involved in early 3T3-L1 preadipocyte differentiation[J]. Journal of Biological Chemistry,2018,293(36):14012-14021. doi:10.1074/jbc.RA118.004401.

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