A 14-amino acids deletion in BmShadow results to non-moult on the 2nd instar in the bivoltine silkworm, Bombyx mori

Juan Zhu  ¹ , Yan-Rong Chen  ² , Tao Geng  ³ , Shun-Ming Tang  ¹ , Qiao-Ling Zhao  ¹ , Xing-Jia Shen  ⁴ Affiliations

• PMID: 33516793
• DOI: 10.1016/j.gene.2021.145450

Abstract

The Bombyx mori Shadow gene (BmShadow) belongs to the superfamily of cytochrome P450 genes. To elucidate the function of the BmShadow gene and its association with diapause, we employed the CRISPR/Cas9 system to knock out the BmShadow gene in the bivoltine strain Qiufeng. The mutant (BmShadow^-/-) was obtained in G₂, exhibiting a 42-base deletion corresponded exactly to the amino acids regions from positions 155 to 168. The larvae of BmShadow^-/- cannot moult at the pre-moulting stage of the 2nd instar. When the BmShadow^-/- larvae were fed with 20E analogue at the late stage of the 2nd instar, they were rescued and developed into the 3rd instar. Rescue experiments indicated that the 20E concentration of BmShadow^-/- larvae was significantly lower than that in WT larvae, and the 20E concentration of BmShadow^-/- larvae which fed 20E analogue was restored to normal levels. Interestingly, the BmShadow^-/- larvae could not moult on the 1st instar when they hatched from eggs after being stored at 5 °C for 40 days or after hibernation, suggesting that the 20E transported from the mother was partially consumed in the diapause maintenance phase. Our study confirmed that BmShadow is involved in 20E synthesis and a 14-amino acids region from position 155 to 168 was essential for its function, also there appears to be no other compensation pathway in vivo, which offered an important potential target locus for the control of silkworm development and the biological control of agricultural and forestry pests.

Keywords: 20-hydroxyecdysterone; BmShadow; Bombyx mori; CRISPR/Cas9; Microinjection.