Plant Gene Expression Quantification

The RNA was extracted using HiPure Plant RNA Kits (Magen), and HiScript^® II Q Select RT SuperMix (Vazyme) was used for cDNA synthase. Gene expression was assessed by real-time PCR using AceQ^® qPCR SYBR^® Green Master Mix (Vazyme) and Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. Primers MBF1c-YG1-UP (5’-GTTAACGCGGCTCTCAGAAG-3’) and MBF1c YG1-DW (5’-TCCTCCAGCTTCTTCGTGTT-3’) were used for BocMBF1c; Tubulin8 was used as an internal control with primers Tubulin-F (5’-CTTCTTTCGTGCTCATTTTGCC-3’) and Tubulin-R (5’-CCATTCCCTCGTCTCCACTTCT-3’) [19 (link)]. The qPCR conditions: initial denaturation of 95 °C for 5 min, then 40 cycles of 95 °C for 10 s followed by 60 °C for 30 s; melting curves were obtained by gradually increasing the temperature from 60 °C to 95 °C for 15 s at a rate of 0.5 °C /s. Relative expression was calculated by the ∆∆Ct threshold method [24 (link)]. For heat and cold treatment, semi-quantitative RT-PCR was used to detect the expression pattern of BocMBF1c with Recombinant Taq DNA Polymerase TaKaRa Taq™ (Takara) and the same primers of qPCR. Semi-quantitative RT-PCR conditions: initial denaturation of 94 °C for 2 min, then 26 cycles for Tubulin8 and 30 cycles for BocMBF1c of 95 °C for 30 s, 56 °C for 15 s, 72 °C for 10 s. Three biological replicates with triple technical replicates were used for all samples.