BmPriS promotes silk gland growth by regulating endoreplication in silkworm

Endoreplication, also known as the endocycle, is a variant of the cell cycle that occurs in the silk glands of silkworms. Although the primase α subunit (PriS) has been reported to be involved in the initiation of DNA replication, its role in silk gland cell endoreplication remains unclear. In our study, we observed that BmPriS expression in the posterior silk gland (PSG) progressively increased during the late fifth instar. Using the CRISPR/Cas9 system, we specifically mutated BmPriS in the PSG, resulting in a decrease in cocoon shell weight and thinner silk fibers. Comparison with the wild-type revealed that the PSG was completely absent in ∆BmPriS silkworms. Immunofluorescence staining revealed a significant reduction in the size of silk gland cells. The expression of the fibroin genes (FibH, FibL, and P25) was nearly silent, whereas that of the sericin genes (Ser1, Ser2, and Ser3) was significantly downregulated. Moreover, EdU staining indicated a marked impairment of endoreplication in PSG cells, accompanied by a significant downregulation of endoreplication-associated minichromosome maintenance genes (MCM3, MCM5, MCM6, and MCM7). Cell cycle- and growth-related genes (CDK2, CyclinE, and Yki) were also significantly downregulated, whereas apoptosis-related genes (Fadd, Daxx, and Dredd) were significantly upregulated. Collectively, these findings indicate that BmPriS regulates silk gland growth and reduces silk production by modulating PSG endoreplication, and by interfering with the cell cycle and apoptosis processes. This study offers novel perspectives on the involvement of PriS in silk gland development.