Transient Protoplast Transformation and Dual-Luciferase Assay

PEG-Ga solution (40% PEG 4000, 0.2 M Mannitol, 0.1 M GaCl₂) was used by protoplasts transformation. The single sample needed 110 μL PEG-Ga solution, and this transformation reaction lasted six minutes before dilution with 440 μL MMG (15 mM MgCl_2, 0.4 M Mannitol, 4 mM MES). Protoplasts after transformation reaction were washed by WI (0.5 M Mannitol, 4 mM MES, 20 mM KCl) for dual-Luciferase assay. The C4-NADP-ME (Zm00001eb121470) promoter sequence was recombined with the PBI221 vector, in which the GUS (β-glucuronidase) reporter gene was replaced with the LUC (fireflyluciferase) reporter gene [43 (link)]. The coding sequences of bHLH157 and NF-YC2 were cloned into the PBI221 vector, and the two recombinant vectors were co-transformed into maize leaf protoplasts to evaluate the relative activity of LUC. Dual-luciferase activity (Promega) was measured to verify the interactions between bHLH157 and/or NF-YC2 and C4 NADP-ME. GUS activity was used as an internal reference to normalize the transformation efficiency of protoplasts, and the relative ratio of LUC/GUS (4 h–0 h) between the experimental group and control was used to represent the relative activity of the promoter [44 (link)].