The ebony Gene in Silkworm Black Pupae Significantly Affects 30 K Proteins During the Pupal Stage

<b>Background/Objectives</b>: The body color and patterns of insects play important roles in foraging, evading predators, mating, thermoregulation, and environmental adaptation. During the rearing of the QiufengN silkworm strain, a mutant with black pupal cuticle (QiufengNBP) was discovered. Preliminary map-based cloning and sequence analysis indicated that the <i>ebony</i> gene might significantly influence the formation of the black pupa mutant and the expression of 30K proteins. This study aims to determine the function of the <i>ebony</i> gene and its effect on the expression of the 30K protein during the pupal stage; <b>Methods and Results</b>: We employed CRISPR/Cas9 gene-editing technology to knock out the <i>ebony</i> gene in the Nistari strain, resulting in individuals with black pupae, named Nistari Black Pupa (NisBP). This confirmed that the <i>ebony</i> gene plays a crucial role in black pupa formation. Two-dimensional electrophoresis (2-DE) analysis of the pupal cuticle of NisBP and its wild-type Nistari found that the <i>ebony</i> gene has a significant impact on the expression of 30K proteins, which are vital for embryonic development and serve as key storage proteins; <b>Conclusions</b>: This study is the first to demonstrate that the <i>ebony</i> gene affects the expression of 30K proteins, laying the foundation for further research on their functions and providing insights into the developmental mechanisms of silkworms.