如皋黄鸡CEBPA基因克隆、生物信息学分析及表达载体构建  

Cloning,Bioinformatics Analysis and Expression Vector Construction of CEBPA Gene in Rugao Yellow Chicken

作　　者：孔瑞红 谢可 王旭 邬晗[1,2] 刘吉英 王颖洁 KONG Ruihong;XIE Ke;WANG Xu;WU Han;LIU Jiying;WANG Yingjie(Jiangsu Key Laboratory of Sericulture Biology and Biotechnology,School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang 212100,China;Key Laboratory of Silkworm Genetic Improvement,Ministry of Agriculture and Rural Affairs,Institute of Sericulture,Chinese Academy of Agricultural Sciences,Zhenjiang 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏省蚕桑与畜禽生物技术重点实验室,镇江212100 [2]农业农村部蚕桑遗传改良重点实验室,中国农业科学院桑蚕科学研究中心,镇江212100

出　　处：《中国畜牧兽医》2024年第12期5185-5197,共13页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(32202660);江苏省研究生科研与实践创新计划项目(SJCX24_2572);江苏省大学生创新训练项目(202310289016Y)。

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�【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

关 键 词：如皋黄鸡 CEBPA基因 生物信息学 表达载体构建 

分 类 号：S831.2[农业科学—畜牧学]