Wheat Stress-Responsive Gene Expression

Wheat seedlings at the two leaf stage that were grown under normal condition were sampled and frozen in liquid nitrogen for 10 min, and then transferred into −80 °C refrigerator for saving. Total RNA was extracted from wheat leaves using an RNAprep plant kit (TIANGEN) and reverse transcribed into cDNA using a PrimeScript First-Strand cDNA Synthesis kit (Takara). SYBR master mix (TIANGEN) was used for qRT-PCR. qRT-PCR was performed following the methods described in Liu et al.60 using an ABI Prism 7500 real-time PCR system (Lifetech). Wheat actin, as an internal reference, was used to normalize all data. Three biological replications for each line were performed in each test. The relative transcript levels of stress-responsive genes (TaMYB32, TaWRKY2, TaWD40, TaCDPK3, TaGST, TaRAB18, TaLEA, TaDHN and TaERF3) were calculated using the 2^−ΔΔCT method61 (link). All primers for the stress-responsive genes are listed in Table S6.