关键词: 家蚕, DNA甲基化, 翅, 细胞自噬, 发育调控

Abstract: 【Aim】This study aims to investigate whether DNA methylation affects wing development of Bombyx mori through regulating autophagy. 【Methods】B. mori ovarian Bm12 cells and B. mori prepupae were treated with 5azadC, a DNA methylation inhibitor, at the doses of 1 and 2 μg, respectively, the number of Bm12 cells was observed under fluorescence microscopy, the methylation level in Bm12 cells was detected by dot blotting, the autophagy intensity was detected by lysosome staining, the expression levels of autophagyrelated protein (Atg) genes were detected by RT-qPCR, and the typing of the autophagy marker LC3 protein in Bm12 cells was detected by Western blotting and immunohistochemistry. Meanwhile, the wing phenotype of adult was observed, and the abnormal wing rate and wing area were calculated. After B. mori prepupae were treated with the autophagy activator SMER28 (2 μg), and using autophagy inhibitor Spautin-1 (2 μg) to rescue 5-aza-dC-treated prepupae, the autophagy intensity in the wing cells was detected by lysosome staining, the wing phenotype was observed, and the abnormal wing rate and wing area were calculated. 【Results】Treatment with 1 μg of 5-aza-dC inhibited the growth and decreased the methylation level of Bm12 cells, increased the autophagy level in Bm12 cells at 12, 24 and 48 h after treatment, and up-regulated the expression of Atg genes in Bm12 cells at 48 h after treatment. Injection of 2 μg of 5-aza-dC into B. mori prepupae increased the autophagy level, up-regulated the expression of Atg genes in adult wing cells, and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 72.62%) and the decreased wing area (decreased by 66%) of adult. Injection of 2 μg of SMER28 into B. mori prepupae increased the autophagy level in the adult wing cells and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 75.13%) and the decreased wing area (decreased by 48.79%) of adult. In addition, rescue experiment using Spautin-1 to rescue the 5-aza-dC-treated prepupae revealed that the autophagy inhibition could alleviate the effect of DNA demethylation on the adult wing development. 【Conclusion】The results of this study demonstrate that DNA methylation plays a role in the wing development of B. mori by regulating autophagy. Our results provide the experimental evidence for the regulation of DNA methylation on the insect development.

Key words: Bombyx mori, DNA methylation, wing, autophagy, developmental regulation