Mechanism of reduced insecticidal protein expression in Bt cotton under low-iron stress based on transcriptome analysis

To provide a theoretical basis for the safety of insecticidal efficiency in Bt cotton, this study investigated the effects of low iron on Bt insecticidal protein content and the physiological and molecular mechanisms in Bt cotton. Transgenic Bt cotton cultivar Zhongmian 50 was grown under low iron (LI, 0.1 μmol L-1 Fe(II)-EDTA) and normal iron (CK, 20.0 μmol L-1 Fe(II)-EDTA) conditions in hydroponic culture to assess the impact of low iron stress on Bt insecticidal protein content, activities of nitrogen metabolism-related enzymes, and substances in cotton seedlings. Differentially expressed genes (DEGs) were screened, and their relative expression patterns were analyzed by high-throughput sequencing, with results verified by qRT-PCR. The results showed that, compared with the control, the insecticidal protein contents significantly decreased under low iron conditions, with a more pronounced reduction in roots than in leaves. Iron deficiency decreased NH4+-N and NO3--N levels in leaves and reduced soluble protein and nitrogen contents in both roots and leaves. Significant reductions in the activities of nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) were observed when Bt insecticidal protein contents in roots and leaves were significantly reduced. High-throughput sequencing identified 11,661 DEGs in roots and 8972 in leaves, with 1652 genes down-regulated in both organs. GO annotation revealed that the functions of the differential genes in two organs were mainly concentrated in response to stimulus, cell wall, plasma membrane, binding, and catalytic activity. KEGG enrichment analysis demonstrated significant changes in phenylpropanoid biosynthesis, phenylalanine metabolism, cysteine and methionine metabolism, plant hormone signal transduction, zeatin biosynthesis, nitrogen metabolism, starch and sucrose metabolism, alanine, aspartate and glutamate metabolism, and tyrosine metabolism in both roots and leaves. Genes coding for NR, NiR1, and GLT1 related to the nitrogen reduction and assimilation pathway were significantly down-regulated under iron deficiency treatment. Therefore, low iron stress inhibits the transcription levels of genes related to nitrogen metabolism in Bt cotton, weakens the physiological activities of nitrogen metabolism, and subsequently reduces Bt insecticidal protein.