Abstract: 【Aim】 This study aims to further explore the possible role and mechanism of G-quadruplex (G4) structure in the regulation of the embryonic development of Bombyx mori by searching for the binding protein of the G4 structure of the embryonic development factor (EDF) gene BmEDF of B. mori. 【Methods】 Circular dichroism (CD) and electrophoretic mobility shift assay (EMSA) were performed to verify whether G4 sequence formed G4 structure in vitro. The effect of the G4 structure of the BmEDF promoter on the regulation of BmEDF expression was verified by the promoter activity experiment. qRT-PCR was performed to determine the expression levels of BmEDF in B. mori embryo at different developmental stages. The protein that could bind to the G4 structure of BmEDF was analyzed by EMSA combined with mass spectrometry. Then, the two candidate proteins BmeIF4H and BmADDH binding with G4 structure were cloned, expressed and purified, respectively. The binding of the candidate proteins BmeIF4H and BmADDH to the G4 structure of BmEDF was verified by EMSA. 【Results】 CD and EMSA experiments demonstrated that the G4 sequence of BmEDF could form G4 structure in vitro. Promoter activity experiments showed that the presence of the G4 structure of BmEDF had a positive regulatory effect on the transcriptional expression of BmEDF. The qRT-PCR results showed that the expression level of BmEDF was significantly increased at 120 h after oviposition. The recombinant BmeIF4H and BmADDH were obtained by prokaryotic expression and purification. EMSA experiments showed that the recombinant BmeIF4H bound to the G4 structure of BmEDF in vitro, while BmADDH did not bind to the G4 structure of BmEDF. 【Conclusion】 The BmeIF4H protein in B. mori embryo may bind to the G4 structure of BmEDF. This study provides the experimental evidence for analyzing the regulatory mechanism of DNA advanced structure in the embryonic development of B. mori.

Key words: Bombyx mori; embryonic development, DNA advanced structure, G-quadruplex, binding protein