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

Abstract

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

CLC Number:

S831.2

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.

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

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