Consistency, distinction, and potential metabolic crosstalk of nitrogen mobilization-related genes in silk production and silk gland biology.

The domesticated silkworm ( ) has evolved a highly efficient nitrogen utilization system to support silk production. The silk glands play a pleiotropic role in sequestering nitrogen resources for silk synthesis, mitigating aminoacidemia by assimilating free amino acids, and reallocating nitrogen during metamorphosis through programmed cell death. However, the specific functions of nitrogen metabolism-related genes in this process remain unclear. Using CRISPR/Cas9-based gene editing, mutations were generated in glutamine synthetase ( ), glutamate synthetase ( ), asparagine synthetase ( ), glutamate dehydrogenase ( ) and glutamate oxaloacetate transaminase 1 ( ). Disruption of , , and consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription, whereas mutation had no such effect. mutants exhibited abnormally enlarged silk glands, whereas and mutants showed delayed programmed cell death in the silk glands. In contrast, mutants displayed normal silk gland morphology but were consistently smaller. Disruption of , , and led to more extensive transcriptional changes, including altered expression of transcription factors in the silk glands, compared with mutants. Both and mutants exhibited up-regulation of and , while only mutants displayed elevated AS enzymatic activity, suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis. mutants showed significantly elevated GOT activity and up-regulation of several metabolic pathways, indicating that AS may functionally interact with GOT in regulating both silk gland development and programmed cell death during metamorphosis.