如皋黄鸡CEBPA基因克隆、生物信息学分析及表达载体构建

doi:10.16431/j.cnki.1671-7236.2024.12.007

摘要/Abstract

摘要： 【目的】试验旨在探究CCAAT/增强子结合蛋白α(CCAAT/enhancer binding protein alpha，CEBPA)基因在如皋黄鸡中的生物学特征、组织表达水平，并构建其表达载体。【方法】对如皋黄鸡CEBPA基因CDS区进行克隆并测序，与其他物种进行相似性比对及系统进化树构建，通过生物信息学软件对如皋黄鸡CEBPA蛋白的理化性质、磷酸化位点、糖基化位点、功能结构域、互作蛋白及二级结构、三级结构进行预测。利用实时荧光定量PCR技术检测CEBPA基因在如皋黄鸡心脏、肝脏、肺脏、肾脏、十二指肠、空肠、腺胃、肌肉、睾丸中的表达情况。构建CEBPA基因过表达(OE-CEBPA)和干扰载体(CEBPA-sh12，CEBPA-sh167，CEBPA-sh727)，并检测各载体活性。【结果】如皋黄鸡CEBPA基因CDS区序列全长975 bp，共编码324个氨基酸。相似性比对结果显示，如皋黄鸡与日本鹌鹑、鸿雁、野鸽、野鸭的CEBPA氨基酸序列相似性均＞94%，其中与日本鹌鹑的相似性最高，达99.4%；系统进化树结果表明，如皋黄鸡与日本鹌鹑的亲缘关系较近，与哺乳动物的亲缘关系较远。如皋黄鸡CEBPA蛋白为亲水不稳定蛋白，存在25个磷酸化位点，含9个N-糖基化位点和109个O-糖基化位点；CEBPA蛋白具有1个转录激活区和1个与DNA结合的亮氨酸拉链结构bZIP；如皋黄鸡CEBPA蛋白二级结构主要由193个无规则卷曲、93个α-螺旋、25个延伸链和13个β-转角组成，三级结构预测结果与二级结构基本一致；CEBPA蛋白主要与TRIB1、PPARG、RXRA、MAPK9、MAPK10、JUN、ESR1等蛋白互作。CEBPA基因在如皋黄鸡各组织中均有表达，其中以肝脏中表达量最高。成功构建CEBPA过表达载体和3个干扰载体，将它们分别转染至鸡成纤维细胞，与对照组相比，过表达组(OE-CEBPA)CEBPA基因表达量显著升高(P＜0.05)，干扰组(CEBPA-sh167)CEBPA基因表达量显著下降(P＜0.05)。【结论】试验获得如皋黄鸡CEBPA基因完整CDS区并成功构建与筛选出具有过表达/干扰活性最佳的鸡CEBPA表达载体，该基因在如皋黄鸡各组织中广泛表达，其中以肝脏中相对表达量较高。结果为进一步研究鸡CEBPA的功能提供了参考依据。

关键词: 如皋黄鸡; CEBPA基因; 生物信息学; 表达载体构建

Abstract: 【Objective】 The aim of this experiment was to investigate the biological characteristics and tissue expression level of CCAAT/enhancer binding protein alpha (CEBPA) gene in Rugao Yellow chicken,and obtain its expression vector.【Method】 The CDS region of CEBPA gene of Rugao Yellow chicken was cloned and sequenced,homology comparison between Rugao Yellow chicken and other species was performed,and phylogenetic tree was constructed.The physicochemical properties,phosphorylation sites,glycoylation sites,functional domains,secondary and tertiary structures,interacting proteins of CEBPA protein of Rugao Yellow chicken were predicted by bioinformatics software.The expression of CEBPA gene in the heart,liver,lung,kidney,duodenum,jejunum,glandular stomach,muscle and testis of Rugao Yellow chicken was detected by Real-time quantitative PCR.Finally,the overexpression vector (OE-CEBPA) and interference vectors(CEBPA-sh12，CEBPA-sh167 and CEBPA-sh727) of CEBPA gene were constructed,and the activities of each vector were detected.【Result】 The CDS region of CEBPA gene in Rugao Yellow chicken was 975 bp in length,encoding 324 amino acids.Homology comparison results showed that the amino acid sequence similarity of CEBPA in Rugao Yellow chicken with Coturnix japonica,Anser cygnoides,Columba livia and Anas platyrhynchos was more than 94%,with the similarity with Coturnix japonica was the highest (99.4%).The phylogenetic tree results showed that Rugao Yellow chicken was closely related to Coturnix japonica,but far related to mammals.The CEBPA protein of Rugao Yellow chicken was hydrophilic and unstable,with 25 phosphorylation sites,including 9 N-glycosylation sites and 109 O-glycosylation sites.CEBPA protein had a transcriptional activation region and a DNA-binding leucine zipper structure bZIP.The secondary structure of CEBPA protein of Rugao Yellow chicken was mainly composed of 193 random coil,93 alpha helix,25 extension chains and 13 beta turn,and the prediction results of tertiary structure were basically consistent with the secondary structure.CEBPA protein mainly interacted with TRIB1,PPARG,RXRA,MAPK9,MAPK10,JUN,ESR1 and other proteins.CEBPA gene was expressed in all tissues of Rugao Yellow chicken,and the expression was the highest in liver.The CEBPA overexpression vector and three interfering vectors were successfully constructed and transfected into chicken fibroblasts,respectively.Compared with the control group,the CEBPA gene expression was significantly increased in the OE-CEBPA group (P＜0.05),and significantly decreased in the CEBPA-Sh167 group (P＜0.05).【Conclusion】 The complete CDS region of CEBPA gene in Rugao Yellow chicken was obtained in this experiment,and the chicken CEBPA expression vector with the best overexpression/interference activity was successfully constructed and screened.The gene was widely expressed in various tissues of Rugao Yellow chicken,and the relative expression of CEBPA gene was higher in liver.The results provided a reference for further study on the function of CEBPA in chicken.

Key words: Rugao Yellow chicken; CEBPA gene; bioinformatics; expression vector construction

中图分类号:

S831.2

孔瑞红, 谢可, 王旭, 邬晗, 刘吉英, 王颖洁. 如皋黄鸡CEBPA基因克隆、生物信息学分析及表达载体构建[J]. 中国畜牧兽医, 2024, 51(12): 5185-5197.

KONG Ruihong, XIE Ke, WANG Xu, WU Han, LIU Jiying, WANG Yingjie. Cloning,Bioinformatics Analysis and Expression Vector Construction of CEBPA Gene in Rugao Yellow Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5185-5197.

参考文献

[1] RAMJI D P, FOKA P.CCAAT/enhancer-binding proteins:Structure, function and regulation [J].The Biochemical Journal, 2002, 365(Pt 3):561-575.
[2] AVELLINO R, HAVERMANS M, ERPELINCK C, et al.An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation [J].Blood, 2016, 127(24):2991-3003.
[3] GUO L, LI X, TANG Q Q.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.
[4] 岳永起, 华永琳, 贾逸格, 等.牦牛CEBPα基因克隆及在脂肪细胞中的表达模式[J].华北农学报, 2021, 36(6):228-234.YUE Y Q, HUA Y L, JIA Y G, et al.Cloning of yak CEBPα gene and its expression pattern in yak adipocytes[J]. Acta Agriculturae Boreali-Sinica, 2021, 36(6):228-234.(in Chinese)
[5] 郭鹏.奶山羊C/EBPα基因对乳腺上皮细胞脂质代谢调控作用的研究[D].杨凌：西北农林科技大学, 2023.GUO P.Regulation of C/EBPα gene on lipid metabolism in mammary epithelial cells of dairy goats[D].Yangling：Northwest A&F University, 2023.(in Chinese)
[6] YU X, LIU R, ZHAO G, et al.Folate supplementation modifies CCAAT/enhancer-binding protein α methylation to mediate differentiation of preadipocytes in chickens [J].Poultry Science, 2014, 93(10):2596-2603.
[7] YAN J, YANG H, GAN L, et al.Adiponectin-impaired adipocyte differentiation negatively regulates fat deposition in chicken [J].Journal of Animal Physiology and Animal Nutrition, 2014, 98(3):530-537.
[8] GUO L, CHANG Y, SUN Z, et al.Effects of Chinese yam polysaccharide on intramuscular fat and fatty acid composition in breast and thigh muscles of broilers [J].Foods, 2023, 12(7):1-14.
[9] 黄婷玉，宋汶羲，王优爽，等.肠道菌群调控动物肝脏脂质代谢的研究进展[J].黑龙江畜牧兽医，2024，11：21-26.HUANG T Y, SONG W X, WANG Y S, et al.Research progress on intestinal flora regulating lipid metabolism in animal livers[J].Heilongjiang Animal Husbandry and Veterinary Medicine, 2024，11:21-26.(in Chinese)
[10] LUO Z, WANG Y, XUE M, et al.Astragaloside Ⅳ ameliorates fat metabolism in the liver of ageing mice through targeting mitochondrial activity[J].Journal of Cellular and Molecular Medicine.2021，25(18):8863-8876.
[11] RICHARDS M P, POCH S M, COON C N, et al.Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens[J].The Journal of Nutrition, 2003, 133(3):707-715.
[12] 杜振伟, 朱帅鹏, 马向飞, 等.鸡CEBPA基因CDS区克隆、表达及生物信息学分析[J].生物技术通报, 2021, 37(8):203-212.DU Z W, ZHU S P, MA X F, et al.Cloning，expression and bioinformatics analysis of the CDS region of chicken CEBPA gene[J].Biotechnology Bulletin, 2021, 37(8):203-212.(in Chinese)
[13] 张燕萍, 李柯晔, 王中亮, 等.鸡肝细胞中C/EBPα基因的敲低及对糖脂代谢相关基因表达的影响[J].中国家禽, 2020, 42(4):15-19.ZHANG Y P, LI K Y, WANG Z L, et al.Knock-down of C/EBPα gene expression in chicken hepatocytes and its effect on the expression of lipid metabolism related genes[J].China Poultry, 2020, 42(4):15-19.(in Chinese)
[14] EICHLER J.Protein glycosylation[J].Current Biology, 2019, 29(7):R229-R231.
[15] PRADÈRE J, HERNANDEZ C, KOPPE C, et al.Negative regulation of NF-κB p65 activity by serine 536 phosphorylation [J].Science Signaling, 2016, 9(442):ra85.
[16] JEONG J, LI Y, ROCHE K W.Camkii phosphorylation regulates synaptic enrichment of shank3[J].ENeuro, 2021, 8(3):0481-20.
[17] LOU F, SUN Y, XU Z, et al.Excessive polyamine generation in keratinocytes promotes self-RNA sensing by dendritic cells in psoriasis [J].Immunity, 2020, 53(1):204-216.
[18] TORCAL GARCIA G, KOWENZ-LEUTZ E, TIAN T V, et al.Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity [J].eLife, 2023, 12(6)：1-33.
[19] HOU C, LU S, SU Y, et al.C/EBP-α induces autophagy by binding to Beclin1 through its own acetylation modification in activated hepatic stellate cells [J].Experimental Cell Research, 2021, 405(2):112721.
[20] 路子显, 常团结, 刘翔, 等.植物碱性亮氨酸拉链(bZIP)蛋白的研究进展(一)——结构、分类、分布和同源性分析[J].遗传, 2001, 6:564-570.LU Z X, CHANG T J, LIU X, et al.Advances in the studies of plant basic leucine Zipper (bZIP) proteins (A)—Structure classification distribution and analyses of homology [J]. Genetics, 2001, 6:564-570.(in Chinese)
[21] SATOH T, KIDOYA H, NAITO H, et al.Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages [J].Nature, 2013, 495(7442):524-528.
[22] LEHRKE M, LAZAR M A.The many faces of PPARγ[J].Cell, 2005, 123(6):993-999.
[23] GOLDSTEIN J T, BERGER A C, SHIH J, et al.Genomic activation of PPARG reveals a candidate therapeutic axis in bladder cancer [J].Cancer Research, 2017, 77(24):6987-6998.
[24] YAN H, OPACHALOEMPHAN C, CARMONA-ALDANA F, et al.Insulin signaling in the long-lived reproductive caste of ants [J].Science, 2022, 377(6610):1092-1099.
[25] WANG Y, BI Y, ZUO Q, et al.MAPK8 regulates chicken male germ cell differentiation through JNK signaling pathway [J].Journal of Cellular Biochemistry, 2018, 119(2):1548-1557.
[26] ALVES-BEZERRA M, COHEN D E.Triglyceride metabolism in the liver[J].Comprehensive Physiology, 2017，8(1)：1-8.
[27] RICHARDS M P, POCH S M, COON C N, et al.Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens[J].The Journal of Nutrition, 2003, 133(3):707-715.
[28] ALVARENGA R, ZANGERONIMO M, PEREIRA L, et al.Lipoprotein metabolism in poultry[J].World’s Poultry Science Journal，2011，67(3)：431-440.
[29] QUIROZ-FIGUEROA K, VITALI C, CONLON D M, et al.TRIB1 regulates LDL metabolism through CEBPα-mediated effects on the LDL receptor in hepatocytes [J].Journal of Clinical Investigation, 2021, 131(22):1-11.