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

The silk-producing lepidopteran silkworm Bombyx mori, has evolved an efficient nitrogen motivation system during domestication. The silk glands of B. mori are pleiotropic in recruiting nitrogen resources for silk production, preventing amino-acidaemia through fixing free amino acids, and facilitating nitrogen reallocation during metamorphosis through programmed cell death. However, the roles of nitrogen motivation related genes in this pleiotropic process remain unclear. Here we generated mutations in glutamine synthetase (GS), glutamate synthetase (GOGAT), asparagine synthetase (AS), glutamate dehydrogenase (GDH) and glutamate oxaloacetate transaminase (GOT1) through CRISPR/Cas9-based gene editing. While consistently decreasing silkworm cocoon and pupa weight, disruption of GS, GOGAT and AS caused significant decreased transcription of silk protein genes but GOT1 did not. GOGAT mutants exhibited abnormally enlarged silk glands, while mutants of GS and AS showed delayed programmed cell death in the silk glands. In contrast, GOT1 mutants displayed normal silk gland biology, albeit with consistently smaller in size. Disruption of GS, GOGAT and AS led to significant changes in the expression of more genes, including transcriptional factors in the silk glands than that of GOT1 did. Mutants of GS and GOGAT both showed up-regulation of AS and GDH but only GOGAT mutants exhibited elevated AS activity, suggesting that GOGAT may complete over AS in nitrogen resource glutamine in the silk glands for silk protein synthesis. Mutants of AS exhibited significantly elevated GOT activity and up-regulation of several metabolism pathways, suggesting that AS may compete with GOT in regulating both silk gland development and programmed cell death during metamorphosis.