鸡CEBPA基因CDS区克隆、表达及生物信息学分析

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

摘要/Abstract

摘要：

为了研究增强子结合蛋白α（CCAAT/Enhancer Binding Protein Alpha,CEBPA）的氨基酸序列特征,以及其编码基因在固始鸡不同组织中的mRNA表达谱。采用PCR技术克隆得到CEBPA的CDS序列全长975 bp,可编码氨基酸325个。生物信息学分析结果显示CEBPA不存在跨膜结构和信号肽,蛋白结构主要由无规则卷曲和α-螺旋结构构成,且多分布在细胞核中;蛋白互做网络构建分析表明,该蛋白与主要与TRBs家族、Runx转录因子家族等相互作用;氨基酸序列对比发现,鸡和鹌鹑的亲缘关系较近;鸡CEBPA 5'调控区-2 000 bp序列上启动子和CpG岛预测发现,该序列上有13个不同的启动子和一个长度1 640 bp的CpG岛;qRT-PCR表明,CEBPA在12周龄固始鸡不同组织中胸肌和腿肌的表达量显著低于其他组织（P<0.05）;不同发育时期胸肌中22周龄的表达量显著低于6周龄和55周龄的表达量（P<0.05）;且CEBPA在罗斯鸡胸肌的表达量显著高于固始鸡胸肌的表达量（P<0.05）。以上结果为进一步深入探讨鸡CEBPA在胸肌的脂质沉积过程中的生物学功能奠定了基础。

关键词: 固始鸡, CEBPA, 生物信息学分析, 胸肌, 表达分析

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

杜振伟, 朱帅鹏, 马向飞, 李东华, 孙桂荣. 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 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.

图/表 7

表1 基因引物序列

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

图 1 鸡 CEBPA CDS 区克隆结果 A：鸡 CEBPA 保守序列 PCR 扩增结果;B：阳性菌落测序结果与 NCBI 上 CEBPA 的 CDS 序列比对结果

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

图 2 CEBPA 蛋白结构和功能的预测 A：鸡 CEBPA 蛋白的跨膜结构域;B：鸡 CEBPA 蛋白的信号肽;C：鸡 CEBPA 蛋白的二级结构;D：鸡 CEBPA 蛋白的三级结构;E：鸡 CEBPA 蛋白互作网络

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

图 3 CEBPA 氨基酸序列保守性分析 A：不同物种的 CEBPA 氨基酸序列的同源性对比;B：不同物种的 CEBPA 系统进化树分析

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

图 4 CEBPA 启动子和CpG 岛预测结构示意图 A：CEBPA 启动子预测示意图;B：EMBOSS 软件预测的甲基化 CpG 岛图谱;C：MethPrimer 软件预测的甲基化 CpG 岛图谱

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

图 5 CEBPA 在固始鸡中的时空表达谱小写字母不同表示不同组织之间比较差异显著（P<0.05）,下同

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

图 6 CEBPA 在 6 周龄固始鸡和罗斯鸡胸肌中的相对表达量比较

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

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