A mutation in the Bombyx mori BmSPI51 gene results in alterations protein abundance in cocoon and unveils compensatory mechanism of silk gland proteinase inhibitors

Abstract

Proteinase inhibitors constitute a significant component of Lepidoptera cocoon. BmSPI51 is the most abundant protease inhibitors in silkworm cocoons. In this study, we aimed to elucidate the biological function of BmSPI51 in both the silkworm silk gland and cocoon formation. To achieve this, we utilized CRISPR/Cas9 gene editing to target the BmSPI51 gene, resulting in premature termination of translation at the 33rd amino acid residue. The mutation of BmSPI51 did not affect the growth and development. Nevertheless, it led to a substantial decrease in the economic traits of silkworm cocoons. Proteomic analysis indicated the abundance of fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), and fibrohexamerin (P25) decreased significantly in the homozygous mutants. Further analysis of cocoon proteins found that the mutants significantly increased the secretion of other protease inhibitors in order to deal with the increased environmental stress resulting from the absence of BmSPI51. Surprisingly, homozygous mutant cocoons exhibited an enhanced inhibitory ability against Saccharomyces cerevisiae compared to the WT cocoons. In conclusion, our study provides a valuable insight into the biological function of protease inhibitors and revealed their roles in cocoon formation and potential applications in biotechnology.