Functional characterization of GAPDH2 through overexpression and dsRNA-mediated RNA interference in Synechocystis

Glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2) plays a vital role in cell growth, stress responses, and various cellular processes in organisms. However, its functional characterization in cyanobacteria, particularly in Synechocystis sp. PCC 6803, remains largely unexplored, especially concerning its overexpression and RNA interference (RNAi) via double-stranded RNA (dsRNA). This study aimed to investigate the biological role of GAPDH2 in Synechocystis sp. PCC 6803 by cloning its complete coding sequence (SyGAPDH2). The SyGAPDH2 protein comprises 350 amino acids with a molecular weight of 86.480 kDa and an isoelectric point of 5.03. The sequence alignment analysis revealed two conserved domains: NADH (Nicotinamide Adenine Dinucleotide)-quinone oxidoreductase subunit NuoI and NADH-ubiquinone/plastoquinone oxidoreductase chain 6. Similarly, Phylogenetic analysis demonstrated high sequence similarity of 96 % and 94 % with Coliform (Gammaproteobacteria bacterium), respectively. We further explored the functional significance of SyGAPDH2 through overexpression using the PpsbAII+SyGAPDH2 vector and double stranded RNA (dsRNA)-mediated silencing with dsGAPDH2. Overexpression significantly enhanced cell growth, while dsRNA-mediated suppression resulted in reduced cell proliferation, with effects observed 12 h post-treatment and persisting up to 36 h. These findings emphasize the essential regulatory role of SyGAPDH2 in cellular development and stress response. This study contributes to our understanding of GAPDH2 functional importance in cyanobacteria, providing a foundation for future investigations into its subcellular localization, additional functional roles, and broader regulatory mechanisms within cyanobacterial cellular processes.