Analysis of gene expression
Total RNAs of apple shoots under different treatments were extracted
using an OminiPlant RNA kit (CoWin Biosciences) per manufacture’s
instruction. The first-strand cDNA was synthesized using the PrimeScript
First Strand cDNA Synthesis Kit (TaKaRa) per manufacture’s instruction.
Quantitative real-time PCR (qRT-PCR) reaction solutions (20 μl) were
assembled by combining the forward and reverse primers (0.25 μM for
each), template cDNA (50 ng), and SYBR Green PCR Master Mix (10 μl). The
reactions (95 °C, 5 min; 95 °C, 15 s, 60 °C, 1 min, for 40 cycles) were
performed using the iCycler iQ5 Detection System (Bio-Rad). 18S rRNA
served as internal control. The relative gene expression was calculated
using the 2-ΔΔCt methods. Three biological repeats
were performed for each individual experiments. Primers used for qRT-PCR
were listed in Table S2.