中华蜜蜂GABARAP基因克隆、生物信息学分析及原核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2022.01.007

Abstract

Abstract: [Objective] The purpose of this study was to perform cloning, identification and bioinformatics analysis of gamma-aminobutyric acid receptor type associated protein (GABARAP) gene, a member of the Atg8 family of autophagy-related proteins in Apis cerana cerana. Prokaryotic expression was carried out in E. coli BL21 (DE3) competent cell, hoping to lay the foundation for the subsequent exploration of the function of this gene. [Method] Primers were designed with reference to Apis mellifera GABARAP gene sequence in GenBank (accession number:XM_001120069.5). The nested PCR was used to amplify and clone the Apis cerana cerana GABARAP gene. The amino acid sequence similarity, secondary structure and tertiary structure were compared and predicted by bioinformatics software. The prokaryotic expression vector of GABARAP gene was constructed. The GABARAP protein was identified by Western blotting and induced by IPTG and then purified. [Result] The Apis cerana cerana GABARAP gene was amplified by nested PCR. The comparison of amino acid sequence similarity analysis using online BLAST tool showed that the GABARAP of Apis cerana cerana was 100% with that of Apis mellifera, Apis dorsata, Apis florea, Bombus terrestris and Bombus impatiens. The phylogenetic tree analysis of amino acid sequence showed that GABARAP of Apis cerana cerana was most closely related to Apis mellifera and farthest from Caenorhabditis elegans. The results of bioinformatics analysis showed that the CDS region of GABARAP gene fragment size was 354 bp, encoding 117 amino acids, the molecular weight of the protein was 13.99 ku and the theoretical isoelectric point was 9.48. The secondary structure of GABARAP protein was mainly composed of 3 alpha helices, 4 beta sheets and 6 peptide binding sites, the predicted results of secondary and tertiary structures were the same. SDS-PAGE analysis showed that the molecular weight of GABARAP protein induced by IPTG was 35 ku. The results of Western blotting showed that His monoclonal antibody could specifically recognize GABARAP protein. [Conclusion] In this study, the GABARAP gene of Apis cerana cerana was cloned successfully, and the GABARAP protein was obtained and analyzed by bioinformatics, which provided a reference for further study on the role of GABARAP gene and its protein in autophagy.

Key words: Apis cerana cerana; GABARAP gene; gene cloning; bioinformatics; prokaryotic expression

CLC Number:

S893.2

YU Huimin, WU Pengjie, LI Nannan, TAN Jing, XU Shufa, WU Jiangli. Cloning, Bioinformatics Analysis and Prokaryotic Expression of GABARAP Gene in Apis cerana cerana[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 60-69.

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Cloning, Bioinformatics Analysis and Prokaryotic Expression of GABARAP Gene in Apis cerana cerana

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