CRISPR/Cas9-Mediated Knockout of BmGDAP2 in the Silkworm, Bombyx mori: Extended Lifespan and Altered Gene Expression Impacting Developmental Pathways.

Abstract

Ganglioside-induced differentiation-associated protein 2 (GDAP2) is a gene involved in hereditary cerebellar ataxia. At present, little is known about the function of GDAP2 in insects. In this study, BmGDAP2 was detected to be highly expressed in the head, epidermis, midgut, and anterior silk glands of silkworms. We generated a knockout mutant, BmGDAP2 (BmGDAP2^KO), using the CRISPR/Cas9 system. Compared with that of the wild-type, the growth cycle of BmGDAP2^KO larvae was significantly prolonged, while their body size was reduced. Furthermore, we found 149 differentially expressed genes (DEGs) between BmGDAP2^KO and the wild-type, including 106 upregulated and 43 downregulated genes. GO annotation analysis indicated that BmGDAP2 primarily influences structural and molecular activities, as well as catalytic and binding functions. KEGG pathway analysis revealed that the differentially expressed genes were mainly enriched in pathways related to peroxidase activity, hormone synthesis, apoptosis, and longevity regulation. Further investigation focused on candidate genes related to these pathways. We found that the expression levels of MAD2L1, which can inhibit cell proliferation and promote apoptosis, and Aurka-b, which plays a crucial role in cell cycle regulation, were significantly reduced in BmGDAP2^KO silkworms. These changes may interfere with the normal functions of cell division, leading to the prolonged developmental cycle observed in BmGDAP2^KO larvae. Our findings demonstrate that knockout of BmGDAP2 significantly prolongs the life cycle of Bombyx mori by affecting genes related to autophagy, apoptosis, and hormone regulation.