Extended Data Fig. 3: Production and detection of OsECT3 transgenic plants.
a, Production of rice OsECT3 CRISPR-Cas9 mutants. The positions of single guide RNA (sgRNA) in the gene (indicated by black arrows) and decoded sequence mutations of the OsECT3 gene are shown. b, c, Detection of relative mRNA and protein levels in WT and COM-E3-F (OsECT3pro::OsECT3-Flag/osect3) plants by RT-qPCR (b) and immunoblotting with anti-Flag antibody (c), respectively. d, Determination of relative OsECT3 mRNA levels in WT and COM-E3-G (OsECT3pro::OsECT3-GFP/osect3), COM-E3K471R (OsECT3pro::OsECT3K471R-GFP/osect3), and COM-E3K471Q (OsECT3pro::OsECT3K471Q-GFP/osect3) plants by RT-qPCR. e-g, Detection of GFP-targeted OsECT3 (e), OsECT3(K471R/Q) (f, g) protein levels using anti-GFP antibodies in the respective complementary transgenic plants. For all data, bars indicate the mean ± s.d. of three replicates (one-way ANOVA with Tukey’s multiple comparison tests, the significance level was p < 0.05). RT-qPCR used ACTIN as a reference gene, each sample are three biological replicates. Immunoblot analyses show the indicated protein expression of the transgenes in T2 seedlings 14 days after germination. Four independent lines are shown for each construct, anti-actin used as a control.
