A Knockout of the OsGAPDHC6 Gene Encoding a Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Reacts Sensitively to Abiotic Stress in Rice

Jin-Young Kim, Ye-Ji Lee, Hye-Mi Lee, Yoo-Seob Jung, Jiyun Go, Hyo-Ju Lee, Ki-Sun Nam, Jong-Hee Kim, Kwon-Kyoo Kang and Yu-Jin Jung

Gene; 6 April 2025, 16(4), 436; https://doi.org/10.3390/genes16040436

Abstract

Background/Objectives: The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, encoded by OsGAPDHC6, plays a crucial role in glycolysis while participating in various physiological and stress response pathways. Methods: In this study, the expression levels of the OsGAPDHC1 and OsGAPDHC6 genes were investigated over time by treating various abiotic stresses (ABA, PEG, NaCl, heat, and cold) in rice seedlings. Results: As a result, the expression levels of both genes in the ABA-treated group increased continuously for 0–6 h and then de-creased sharply from 12 h onwards. The mutational induction of the GAPDHC6 gene by the CRISPR/Cas9 system generated a stop codon through a 1 bp insertion into protein production. The knockout (KO) lines showed differences in seed length, seed width, and seed thickness compared to wild-type (WT) varieties. In addition, KO lines showed a lower germination rate, germination ability, and germination index of seeds under salt treatment compared to WT, and leaf damage due to 3,3′-diaminobenzidine (DAB) staining was very high due to malondialdehyde (MDA) accumulation. The KO line was lower regarding the expression level of stress-related genes compared to WT. Conclusions: Therefore, the OsGAPDHC6 gene is evaluated as a gene that can increase salt resistance in rice as it actively responds to salt stress in the early stages of growth, occurring from seed germination to just before the tilling stage.

See https://www.mdpi.com/2073-4425/16/4/436

Figure 3: Selection of transgenic plants and gene-edited plants using deep-sequencing analysis. (A) Amplification of bar region and determination of T-DNA insertion by PCR analysis. M, 100 bp marker; PC, positive control; WT, wild type; DW, negative control. (B,C) Plants gene-edited using deep-sequencing analysis. The part highlighted in black represents sgRNA, and the underlined letters represent the PAM (NGG) region. Red letters indicate inserted bases, and dashes indicate deleted bases.