Fastking gdna dispelling rt supermix reverse transcription kit

The expression pattern of potato StMAPKKs genes under abiotic stress was analyzed in the laboratory [52 ], and the results showed that the expression of most of the genes (except StMAPKK3) in MAPKKs was up-regulated under stress treatments, among which StMAPKK5 was up-regulated under drought and salt stress. Therefore, StMAPKK5 was selected as the target gene in this study. The StMAPKK5 gene sequence (ID: Soltu.DM.03G023940.1) was retrieved from the potato database Spud DB (http://spuddb.uga.edu/index.shtml accessed on 13 January 2024). The total RNA extraction and cDNA first strand synthesis of potato cultivar ‘Atlantic’ was performed using the TRNzol Universal Plant Total RNA Extraction Kit and FastKing gDNA Dispelling RT SuperMix Reverse Transcription Kit (Tiangen, Beijing, China), respectively. The CDS region of the StMAPKK5 gene was cloned from the cDNA library of leaves of the potato variety ‘Atlantic’ as a template (Gene CDS sequences are shown in Supplemental Table S1). The primer sequences are shown in Supplementary Table S2. The reaction system was PrimerSTAR HS (Premix) 12.5 μL, ddH₂O 9 μL, cDNA template 1.5 μL, and pCAMBIA1300-35S-StMAPKK5-F/R 1 μL; the reaction conditions were: pre-denaturation: 98 °C for 3 min; denaturation: 98 °C for 10 s, annealing: 55 °C for 5 s, extension: 72 °C for 100 s, cycling 34 times, extension 72 °C for 5 min.

For total RNA extraction and first-strand cDNA synthesis, please refer to the manual of TRNzol Universal Total RNA extraction Kit and FastKing gDNA Dispelling RT SuperMix reverse transcription kit (Tiangen, Beijing, China), respectively. The CDS region of the StCIPK18 gene was cloned using the leaf cDNA of the potato variety “Atlantic” as a template. The reaction system was as follows: OE-StCIPK18-F 1 μL, OE-StCIPK18-R 1 μL, cDNA template 1 μL, PrimerSTAR HS (Premix) (Takara, Dalian, China) 10 μL, ddH2O 7 μL. The reaction conditions were as follows: 95 °C for 5 min, followed by 34 cycles of 95 °C for 30 s, 58.7 °C for 30 s, 72 °C for 90 s and finally, 72 °C for 10 min. The primer sequences are shown in Table S1.