Cloning,Expression and Bioinformatics Analysis of the CDS Region of Chicken CEBPA Gene

doi:10.13560/j.cnki.biotech.bull.1985.2020-1410

Abstract

Abstract:

This work is to investigate the amino acid sequence characteristics of CCAAT/Enhancer Binding Protein Alpha（CEBPA）and the mRNA expression profile of its coding gene in different tissues of Gushi chicken. The CDS sequence of CEBPA with a total length of 975 bp was cloned by PCR,and it encoded 325 amino acids. The bioinformatics analyses showed that there was no transmembrane structure and signal peptide in CEBPA,and the protein structure was mainly composed of irregular crimp and α-spiral,and most of them were distributed in the nucleus. The construction of protein interaction network indicated that CEBPA might interact with TRBs family and Runx transcription factors family. Amino acid sequence comparison found that chicken and quail were closely related and had 13 different promoters and a 1 640 bp CpG island on its -2 000 bp sequence of the 5' regulatory region. qRT-PCR results showed that the expression of CEBPA gene in the breast muscle and leg muscle was significantly lower than that in other tissues of 12-week Guishi chickens（P<0.05）. The expression of CEBPA in the breast muscle of 22-week chicken was significantly lower than that in 6-week and 55-week ones（P < 0.05）. The expression of CEBPA in the breast muscle of Ross chicken was always significantly higher than that in the breast muscle of Gushi chicken（P<0.05）. The above results provide a theoretical basis of further exploring the biological function of chicken CEBPA gene in the lipid deposition process of chicken breast muscle.

Key words: Gushi chicken, CEBPA gene, bioinformatics analysis, breast muscle, expression analysis

DU Zhen-wei, ZHU Shuai-peng, MA Xiang-fei, LI Dong-hua, SUN Gui-rong. Cloning,Expression and Bioinformatics Analysis of the CDS Region of Chicken CEBPA Gene[J]. Biotechnology Bulletin, 2021, 37(8): 203-212.

Figures/Tables 7

Table 1 Gene primer sequences

目的基因Gene name	引物序列 Primer sequence（5'-3'）	用途 Purpose
CEBPA	F：ATGGAGCAAGCCAACTTCTACGAGG R：GGCGCAGCTGCCCATGGCCTTCAC	基因克隆Genetic cloning
CEBPA	F：TTCTACGAGGTCGATTCCCG R：AGCCTCTCTGTAGCCGTAG	基因表达分析Gene expression analysis
β-actin	F：CAGCCAGCCATGGATGATGA R：ACCAACCATCACACCCTGAT	内参基因Endogeneity

Fig.1 Cloning results of chicken CEBPA CDS region A：PCR amplification results of chicken CEBPA conserved sequences;B：sequencing results of positive colonies compared with CDS sequences of CEBPA on NCBI

Fig.2 Prediction of CEBPA protein structure and function A：Transmembrane structural domain of chicken CEBPA protein. B：Signal peptide of chicken CEBPA protein. C：Secondary structure of chicken CEBPA protein. D：Tertiary structure of chicken CEBPA protein. E：Chicken CEBPA protein interaction network

Fig.3 Conserved amino acid sequence analysis of CEBPA A：Comparison of the homology of the amino acid sequences of CEBPA in different species. B：Phylogenetic tree analysis of CEBPA in different species

Fig.4 Schematic diagram of the predicted structure of CEBPA promoter and CpG island A：Schematic diagram of CEBPA promoter prediction. B：Methylation CpG islands predicted by EMBOSS software. C：Methylation CpG islands predicted by MethPrimer software

Fig.5 Spatial and temporal expression profiles of CEBPA in Gushi chickens Different lowercase letters indicate significant differences between different tissues for comparison（P<0.05）,the same below

Fig. 6 Comparison of the relative expression of CEBPA in the pectoral muscle of 6-week-old Gushi and Rose chickens

References 39

[1]	黄明捷, 陈祥, 张勇, 等. CEBPA基因的生物学效应及其在畜禽生产中的研究进展[J]. 中国畜牧杂志, 2020, 56(1):44-50.
	Huang MJ, Chen X, Zhang Y, et al. Biological effects of CEBPA gene and its research progress in animal husbandry[J]. Chinese Journal of Animal Science, 2020, 56(1):44-50.
[2]	Olga K, Anna M, Natalia L. Aberrant expression of alternative isoforms of transcription factors in hepatocellular carcinoma[J]. World Journal of Hepatology, 2018, 10(10):645-661. doi: 10.4254/wjh.v10.i10.645 URL
[3]	Guo L, Li X, Tang QQ. Transcriptional regulation of adipocyte differentiation:a central role for CCAAT/enhancer-binding protein(C/EBP)β[J]. The Journal of Biological Chemistry, 2015, 290(2):755-761. doi: 10.1074/jbc.R114.619957 URL
[4]	Cao H, Wen Y, Xu XL, et al. Investigation of the CEBPA gene expression pattern and association analysis of its polymorphisms with meat quality traits in chickens[J]. Animal Biotechnology, 2020(1):1-9.
[5]	Luo J, Dou L, Yang ZY, et al. CBFA2T2 promotes adipogenic differentiation of mesenchymal stem cells by regulating CEBPA[J]. Biochemical and Biophysical Research Communications, 2020, 529(2):133-139. doi: 10.1016/j.bbrc.2020.05.120 URL
[6]	Moseti D, Regasse A, Kim WK. Molecular regulation of adipogenesis and potential anti-adipogenic bioactive molecules[J]. International Journal of Molecular Sciences, 2016, 17:124. doi: 10.3390/ijms17010124 URL
[7]	Fracaro L, Senegaglia AC, Herai RH, et al. The expression profile of dental pulp-derived stromal cells supports their limited capacity to differentiate into adipogenic cells[J]. International Journal of Molecular Sciences, 2020, 21(8):2753. doi: 10.3390/ijms21082753 URL
[8]	Liuy W, Meridew JA, Aravamudhan A, et al. Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression[J]. Respiratory Research, 2019, 20(1):1-12. doi: 10.1186/s12931-018-0967-9 URL
[9]	Xie L, Zou L, Chen J, et al. All-trans retinoic acid inhibits bone marrow mesenchymal stem cell commitment to adipocytes via upregulating FRA1 signaling[J]. International Journal of Endocrinology, 2020, 2020:6525787.
[10]	王素星, 魏金刚, 邵伟华, 等. 白藜芦醇对成年期追赶生长大鼠体成分的影响及机制[J]. 中国应用生理学杂志, 2020, 36(1):85-89.
	Wang SX, Wei J G, Shao WH, et al. Effect of resveratrol on body composition in adult catch-up growth rats and its mechanism[J]. Chinese Journal of Applied Physiology, 2020, 36(1):85-89.
[11]	Fatima LA, Campello RS, Barreto-Andrade JN, et al. Estradiol stimulates adipogenesis and Slc2a4/GLUT4 expression via ESR1-mediated activation of CEBPA[J]. Molecular and Cellular Endocrinology, 2019, 498:110447. doi: 10.1016/j.mce.2019.05.006 URL
[12]	Gao Y, Sun Y, Duan K, et al. CpG site DNA methylation of the CCAAT/enhancer-binding protein, alpha promoter in chicken lines divergently selected for fatness[J]. Anim Genet, 2015, 46(4):410-417. doi: 10.1111/age.2015.46.issue-4 URL
[13]	Qiu J, Wang W, Hu S, et al. Molecular cloning, characterization and expression analysis of C/EBP α, β and δ in adipose-related tissues and adipocyte of duck(Anas platyrhynchos)[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 2018, 221:29-43.
[14]	周扬. 秦川牛脂肪沉积相关基因筛选及可变剪接对基因表达和细胞定位的影响研究[D]. 杨凌:西北农林科技大学, 2017.
	Zhou Y. Identifying genes related to adipogenesis in qinchuan cattle and studying the effects of alternative splicing on gene expression and cellular localization[D]. Yangling:Northwest A&F University, 2017.
[15]	王梦心, 周玉龙, 贾伟强, 等. 黑龙江省某规模化牧场BVDV病毒分离鉴定及生物学信息分析[J]. 黑龙江八一农垦大学学报, 2020, 32(5):13-22.
	Wang MX, Zhou YL, Jia WQ, et al. Isolation identification and biological information analysis of BVDV virus in a large-scale pasture[J]. Journal of Heilongjiang Bayi Agricultural University, 2020, 32(5):13-22.
[16]	张旻, 林矫矫, 洪炀, 等. 日本血吸虫MBLAC1基因的克隆及生物信息学分析[J]. 中国人兽共患病学报, 2018, 34(4):315-322.
	Zhang M, Lin JJ, Hong Y, et al. Molecular cloning and bioinformatics analysis on MBLAC1 of Schistosoma japonicum[J]. Chinese Journal of Zoonoses, 2018, 34(4):315-322.
[17]	吴亚飞. PPARG通过负调控Six2影响肾透明细胞癌的增殖和凋亡[D]. 重庆:重庆医科大学, 2019.
	Wu YF. PPARG negatively modulates six2 in tumor formation of clear cell renal cell carcinoma[D]. Chongqing:Chongqing Medical University, 2019.
[18]	王锦秋. 基于HNF1α/HNF4α探讨醋柴胡增强大黄酸肝靶向作用[D]. 广州:广东药科大学, 2019.
	Wang JQ. Exploring the effect of enhancing liver-targeting of rein by vinegar-baked radix bupleuri based on HNF1A/HNF4A[D]. Guangzhou:Guangdong Pharmaceutical University, 2019.
[19]	Wang Z, Bishop EP, Burke PA. Expression profile analysis of the inflammatory response regulated by hepatocyte nuclear factor4α[J]. BMC Genomics, 2011, 12(1):128. doi: 10.1186/1471-2164-12-128 URL
[20]	Sung HY, Guan H, Czibula A, et al. Human tribbles-1 controls proliferation and chemotaxis of smooth muscle cells via mapk signaling pathways[J]. The Journal of Biological Chemistry, 2007, 282(25):18379-18387. doi: 10.1074/jbc.M610792200 URL
[21]	侯振林. TRIB2通过AP4/p21信号轴抑制结直肠癌细胞衰老的研究[D]. 武汉:华中科技大学, 2019.
	Hou ZL. TRIB2 inhibits senescence of colorectal cancer cells via AP4/p21 signal axis[D]. Wuhan:Huazhong University of Science and Technology, 2019.
[22]	Zhang P, Iwasaki-Arai J, Iwasaki H, et al. Enhancement of hematopoietic stem cell repopulating capacity and self-renewal in the absence of the transcription factor C/EBPα[J]. Immunity, 2004, 21(6):853-863. pmid: 15589173
[23]	Ye M, Zhang H, Amabile G, et al. C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence[J]. Nature Cell Biology, 2013, 15(4):385-394. doi: 10.1038/ncb2698 URL
[24]	Radomska HS, Huettner CS, Zhang PU, et al. CCAAT/enhancer binding protein α is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors[J]. Mol Cell Biol, 1998, 18(7):4301-4314. doi: 10.1128/MCB.18.7.4301 pmid: 9632814
[25]	Gonzalez D, Luyten A, Barthoidy B, et al. ZNF143 protein is an important regulator of the myeloid transcription factor C/EBPα[J]. Journal of Biological Chemistry, 2017, 292(46):18924-18936. doi: 10.1074/jbc.M117.811109 pmid: 28900037
[26]	马晓阳, 张爽, 江泽友. 非小细胞肺癌中CDO1、RASSF1A和SHOX2基因甲基化的应用[J]. 临床与实验病理学杂志, 2020(10):1175-1179.
	Ma XY, Zhang S, Jiang ZY. Application of CDO1, RASS1A and SHOX2 gene methylation in non-small cell lung cancer[J]. Chin J Clin Exper Pathol, 2020(10):1175-1179.
[27]	Jeschke J, Bizet M, Desmedt C, et al. DNA methylation-based immune response signature improves patient diagnosis in multiple cancers[J]. J Clin Invest, 2017, 127(8):3090-3102. doi: 10.1172/JCI91095 pmid: 28714863
[28]	Liu LM, Sun WZ, Fan XZ, et al. Methylation of C/EBPα by PRMT1 inhibits its tumor-suppressive function in breast cancer[J]. Cancer Research, 2019, 79(11):2865-2877. doi: 10.1158/0008-5472.CAN-18-3211 pmid: 31015230
[29]	Yang X, Wang G, Wang Y, et al. Histone demethylase KDM7A reciprocally regulates adipogenic and osteogenic differentiation via regulation of C/EBPα and canonical Wnt signalling[J]. Journal of Cellular and Molecular Medicine, 2019, 23(3):2149-2162. doi: 10.1111/jcmm.2019.23.issue-3 URL
[30]	王梦岩, 姜平, 余湘, 等. 奶牛乳腺组织中ACSL5基因的表达量差异分析[J]. 中国兽医学报, 2019, 39(1):100-104.
	Wang MY, Jiang P, Yu X, et al. Analysis of the expression level of ACSL5 gene in mammary tissues of dairy cows[J]. Chinese Journal of Veterinary Science, 2019, 39(1):100-104.
[31]	吴国芳, 王磊, 周继平. 小鼠Sirt2基因的组织表达谱及其成肌细胞分化过程中的时序表达[J]. 青海畜牧兽医杂志, 2017, 47(6):27-30.
	Wu GF, Wang L, Zhou JP. Study of mouse Sirt2 gene expression in different tissues and myoblast differentiation stage[J]. Chin Qinghai J Anim Veter Sci, 2017, 47(6):27-30.
[32]	Brown AL, Hahn CN, Scott HS. Secondary leukemia in patients with germline transcription factor mutations(RUNX1, GATA2, CEBPA).[J]. Blood, 2020, 136(1):24-35. doi: 10.1182/blood.2019000937 pmid: 32430494
[33]	Chen C, Qi JQ, Chu TT, et al. Efficacy of haploidentical hematopoietic stem cell transplantation in treatment of intermediate risk acute myeloid leukemia with negative for FLT3-ITD, NPM1 or biallelic CEBPA mutation[J]. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2020, 28(3):731-736.
[34]	Braun TP, Coblentz C, Smith BM, et al. Combined inhibition of JAK/STAT pathway and lysine-specific demethylase 1 as a therapeutic strategy in CSF3R/CEBPA mutant acute myeloid leukemia[J]. PNAS, 2020, 117(24):13670-13679. doi: 10.1073/pnas.1918307117 URL
[35]	郑圣晗, 孔令琳, 朱鹏飞, 等. 不同鸡品种BNK基因序列比较分析[J]. 浙江农业学报, 2017, 29(10):1648-1653.
	Zheng SH, Kong LL, Zhu PF, et al. Comparative analysis of BNK gene sequences in different chicken breeds[J]. Acta Agriculturae Zhejiangensis, 2017, 29(10):1648-1653.
[36]	付守艺, 闫峰宾, 陈毅, 等. 鸡miR-15a对胸肌肌内脂肪沉积的调控作用[J]. 畜牧与兽医, 2018, 50(4):60-65.
	Fu SY, Yan FB, Chen Y, et al. The Function of miR-15a in regulating intramuscular fat deposition in pectoralis muscle in chicken[J]. Animal Husbandry and Veterinary Medicine, 2018, 50(4):60-65.
[37]	蒋可人, 20周龄和55周龄固始母鸡肉质特性及其分子调控基础的研究[D]. 郑州:河南农业大学, 2016.
	Jiang KR. Comparison of meat quality related characteristics between gushi hens at 20 and 55 weeks and research of the basic molecular regulation[D]. Zhengzhou:Henan Agricultural University, 2016.
[38]	张立芳. 加强产蛋期管理提高蛋鸡生产效率[J]. 山东畜牧兽医, 2019, 40(7):23-24.
	Zhang LF. Strengthen the management of laying time and improve the production efficiency of laying hens[J]. Shangdong J Anim Sci Veter Med, 2019, 40(7):23-24.
[39]	刘丽元, 杨彦军, 安克龙, 等. 静原鸡ELOVL2和ELOVL5基因表达的组织特异性研究[J]. 浙江农业学报, 2017, 29(8):1290-1296.
	Liu LY, Yang YJ, An KL, et al. Tissue-specific expression analysis of ELOVL2 and ELOVL5 genes in Jingyuan chicken[J]. Acta Agriculturae Zhejiangensis, 2017, 29(8):1290-1296.