3.5. Experimental Validation of Gene Expression with qRT-PCR

The synthesis of cDNA was performed using FastKing RT Kit (With gDNase) (TIANGEN, Beijing, China). The gene-specific quantitative real-time PCR primers used in this study were documented in Supplementary Table S7. Real-time PCR was performed using a SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China), and was carried out using an Eppendorf Mcep Realplex 4s System (Eppendorf, Hamburg, Germany). Reactions started at 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 25 s, and a melting curve step at 60–95 °C. Each qRT-PCR reaction was performed on three biological replicates. The relative expression levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap3, Synpcc7942_1939), RNA polymerase sigma factor gene (rpoD, Synpcc7942_0649) [92] and phosphoenolpyruvate carboxylase gene (ppc, Synpcc7942_2252) [93], and were calculated using the 2^-∆∆CT method [94].