Consistence, distinction and possible metabolic crosstalk of the nitrogen motivation related genes in affecting silk production and silk gland biology

The domesticated silkworm ( Bombyx mori) 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 ( GS), glutamate synthetase ( GOGAT), asparagine synthetase ( AS), glutamate dehydrogenase ( GDH) and glutamate oxaloacetate transaminase 1 ( GOT1). Disruption of GS, GOGAT, and AS consistently reduced silkworm cocoon and pupal weight and significantly down-regulated silk protein gene transcription, whereas GOT1 mutation had no such effect. GOGAT mutants exhibited abnormally enlarged silk glands, whereas GS and AS mutants showed delayed programmed cell death in the silk glands. In contrast, GOT1 mutants displayed normal silk gland morphology but were consistently smaller. Disruption of GS, GOGAT, and AS led to more extensive transcriptional changes, including altered expression of transcription factors in the silk glands, compared with GOT1 mutants. Both GS and GOGAT mutants exhibited up-regulation of AS and GDH, while only GOGAT mutants displayed elevated AS enzymatic activity, suggesting that GOGAT may compete with AS for glutamine in the silk glands to support silk protein synthesis. AS 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.