Tb green premix ex taq 2 2 tli rnaseh plus kit

Total leaf RNA extraction was performed using a kit (Yueyang Hua, Yueyang, China, Cat:0416-50gk). The first cDNA strand was synthesized using a reverse transcription kit (Tiangen, Beijing, China. Cat:KR118-02). The primer sequences of the related genes were downloaded from the GenBank library of NCBI. The primer design is shown in Table S1. qRT-PCR analysis was performed using the reverse transcription product cDNA as the template and 18S as the internal reference gene. qRT-PCR was performed using the TB Green Premix Ex Taq II (2×) (Tli RNaseH Plus) kit (TaKaRa, Kyoto, Japan) in the CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA). The reaction system was 20 μL, consisting of 9 μL TB Green Premix Ex Taq II (TliRNaseH Plus), 7 μL ddH₂O, 2 μL cDNA template and 1 μL forward and reverse primers. The cycling progression was as follows: 95 °C for 3 min; 95 °C for 10 s and 56 °C for 30 s, for a total of 39 cycles. Gene expression change ploidy analysis was calculated using 2^−ΔΔCt, and relative mRNA expression levels were normalized using 18S. The qRT-PCR validation of the DEGs is shown in Figure S1.

Leaf total RNA extraction was performed using a kit (Yueyang Hua, China). The first strand of cDNA was synthesized using a reverse transcription kit (Tiangen, China). The primer sequences of the relevant genes were downloaded separately from the GenBank library of NCBI. The primer design is shown in Table 4. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using the product cDNA of reverse transcription as a template, and 18S was used as an internal reference gene. The qRT-PCR was tested using the TB Green Premix Ex Taq II (2×) (Tli RNaseH Plus) Kit (TaKaRa, Japan) on a CFX96 Real-Time PCR Detection System instrument (Bio-Rad Laboratories, Hercules, CA, USA). The reaction system was 10 μL, including 5 μL TB Green Premix Ex Taq II (TliRNaseH Plus), 2 μL ddH2O, 1 μL cDNA template, and 1 μL forward and reverse primers, respectively. The reaction procedure was as follows: 95 °C for 3 min; 95 °C 10 s, 56 °C 30 s, a total of 39 cycles. Gene expression change ploidy analysis was calculated using 2−ΔΔCt, and relative mRNA expression levels were normalized using 18S.