关键词: 大豆, GmNIC1, 盐碱胁迫, NAD⁺, NADP⁺

Abstract:

【Objective】Saline-alkali stress is one of the significant abiotic stresses that restrict soybean production. Nicotinamide enzyme gene GmNIC1 is a key enzyme gene involved in the synthesis of NAD⁺ in soybeans. It has been previously established that this gene participates in abiotic stress responses. Investigating the function of GmNIC1 under saline-alkali stress lays the foundation for soybean breeding for saline-alkali tolerance.【Method】Using the soybean cultivar Hefeng 25 as the receptor, GmNICl-OE (overexpression) and GmNICl-KO (knockout) transgenic T₃ generation soybean plants were generated, and their growth and physiological indicators under saline-alkali stress were measured. Transcriptome sequencing was performed on wild-type (WT), GmNICl-OE, and GmNICl-KO plants, followed by GO functional enrichment and KEGG pathway enrichment analysis. A protein-protein interaction network was constructed based on the differentially expressed genes of the three comparative combinations, and proteins interacting with GmNIC1 were identified and validated using quantitative real-time PCR.【Result】After saline-alkali stress, GmNICl-OE plants exhibited yellowing of newly grown leaves but erect petioles and increased plant height; GmNICl-KO plants showed wilted and dried leaves, severed yellowing of stems and near-death: WT plants had yellowish-brown stems and leaves that were less yellowed and wilted than those of the GmNICl-KO plants. Compared to WT and GmNICl-KO plants, GmNIC1-OE plants showed increased NAD⁺ content and decreased NADP⁺ content, leading to glucose accumulation, increased activity of SOD and CAT enzymes, and reduced O₂^- and H₂O₂ content. Transcriptome sequencing revealed a total of 747 differentially expressed genes in the OE-1 vs KO-1 comparative combination, including 622 upregulated and 125 downregulated differentially expressed genes. The number of differentially expressed genes in this combination was significantly higher than that in the other two groups. The functions of differentially expressed genes in the three comparative combinations were mainly concentrated in lactase activity, transcriptional regulation activity, and oxidoreductase activity (acting on NADP (H)); metabolic pathways were mainly involved in the biosynthesis of secondary metabolites, exogenous substance metabolism, glucosinolate biosynthesis, galactose metabolism, MAPK signaling pathway, glutathione metabolism, etc. Four proteins related to GmNIC1 were identified, suggesting that Glyma.04G055000.1 positively interacts with GmNICl, and Glyma.12G086500.1 negatively interacts with GmNIC1.【Conclusion】GmNIC1 increases NAD⁺ content and reduces NADP⁺ content to accumulate glucose and enhance antioxidant enzyme activity to eliminate ROS, thereby strengthening the plant's resistance to salt and alkali stress. The GmNIC1 protein can also collaborate with Glyma.04G055000.1 and Glyma.12G086500.1 to jointly regulate the growth of soybean seedlings under salt and alkali stress.

Key words: soybean, GmNIC1, salt and alkali stress, NAD⁺, NADP⁺