Sybr master mix

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.

Total RNA was isolated from the BM samples of the MDS patients using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and was then reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit (Promega Corporation, Madison, Wisconsin, USA) according to the experimental instructions. The relative expression levels of PDCD1, TIGIT, CD47, and KIR3DL2 were detected by qRT-PCR with SYBR Master Mix (TIANGEN, Beijing, China), and β-actin was selected as an internal control. The primer sequences for qRT-PCR are shown in Table S2. The expression levels of PDCD1, TIGIT, CD47, and KIR3DL2 are presented as 2^−ΔCT.

The maize tissue total RNAs were isolated by using the method was described by Yu et al.^{61 (link)}, and first-strand cDNAs were synthesized using a PrimeScript First Strand cDNA Synthesis kit (TaKaRa, Japan). The cDNAs were combined with SYBR master mix (TIANGEN, China), and an ABI7300 system (Applied Biosystems, USA) was used to monitor the kinetics of the PCR products for qRT-PCR (consist of 94 °C for 3minutes, and then 40 cycles of 94 °C for 30 s, 60 °C for 15 s, 72 °C for 34 s). The maize actin gene (GRMZM2G126190) was used as an internal control for normalization of the template cDNA. The amount of accumulated transcript of the ZmWRKY65 gene normalized to the internal actin control gene was determined using the 2^−ΔΔCT method. The qRT-PCR primers of ZmWRKY65 were provided in Table S2. Each sample PCR was repeated three to four biological replicates.