生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 203-212.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1410
收稿日期:
2020-11-19
出版日期:
2021-08-26
发布日期:
2021-09-10
作者简介:
杜振伟,男,硕士,研究方向:动物遗传育种与繁殖;E-mail: 基金资助:
DU Zhen-wei(), ZHU Shuai-peng, MA Xiang-fei, LI Dong-hua(), SUN Gui-rong
Received:
2020-11-19
Published:
2021-08-26
Online:
2021-09-10
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。
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.
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 |
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
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