关键词: 西方蜜蜂, 核心结合因子β亚基, 基因克隆, 时期表达谱, CRISPR/Cas9

Abstract: 【Aim】Core binding factor (CBF) β subunit (CBFβ) is an important transcription factor that plays a crucial role in insect embryonic development and immune regulation, but it has not been reported in Apis mellifera. This study aims to explore the physiological function of CBFβ in A. mellifera using CRISPR/Cas9 technology. 【Methods】 The coding sequence (CDS) of AmBgb (A. mellifera big brother) was cloned and subjected to bioinformatics analysis. The expression levels of AmBgb in A. mellifera at different developmental stages (1-3-day-old worker eggs, 1-, 3- and 5-day-old worker larvae, worker prepupa, 1-, 3-, 5-, 7- and 9-day-old worker pupa, newly emerged workers, 10-day-old nurses and 21-day-old foragers) were detected using qRT-PCR. CRISPR/Cas9 was used to knock out AmBgb in the eggs of A. mellifera, and the developmental and survival statuses of the eggs were monitored. Target sites were detected through PCR and sequencing to validate the physiological function of AmBgb in A. mellifera.【Results】 The full-length CDS of AmBgb of A. mellifera is 759 bp, encoding 252 amino acids with a CBFβ domain located at amino acids 39-201. The molecular weight of AmBgb was 28 982.37 D. AmBgb had no signal peptide or transmembrane domain, suggesting it functions as an intracellular protein. Phylogenetic tree indicated that AmBgb in A. mellifera was highly homologous to Bgb in A. cerana (with the amino acid sequence identity of 99.21%), forming the closest evolutionary relationship. The expression levels of AmBgb in the 2- and 3-day-old eggs were higher than those in the other developmental stages. Among 43 CRISPR/Cas9-treated eggs of A. mellifera, 5 eggs successfully hatched into larvae without target site mutations of AmBgb. Seven dead eggs were randomly tested, and it was found that the target sites of AmBgb in 6 eggs showed gene editing, including insertions, deletions or substitutions in different lengths. 【Conclusion】AmBgb of A. mellifera was highly expressed in the 2- and 3-day-old eggs, and AmBgb knockout resulted in failure to hatch into larvae. These results indicate that AmBgb plays an important role in the embryonic development of A. mellifera, providing theoretical guidance for studying the physiological function of this gene in other hymenopteran insects.

Key words: Apis mellifera, core binding factor β subunit, gene cloning, stage-specific expression profile, CRISPR/Cas9