Trizol

The total RNA of the abovementioned plant materials was extracted by TRIzol (Coolaber) and 1.2 μg total RNA was reverse-transcribed into cDNA (Vazyme, Nanjing, China) for subsequent experiments. The qRT-PCR experiment was performed using the CFX384 real-time system (Biorad, America, CA, USA). The ChamQ universal SYBR qPCR Master Mix (Vazyme, China) reagent was utilized. Every experiment had three biological duplications. The reference gene was the OsUBQ of Oryza sativa. The primers used in the experiment are listed in Table S2.

The following reagents were used: NG (Shandong Fujiao Group, Donga Town Ejiao Co., Ltd, lot: 15060022); vinorelbine (Beijing Century Aoke Biotechnology Co., Ltd., lot: 170410); chloramphenicol (Genvien, USA, lot: 6428010150); methotrexate (China Food and Drug Control Institute, lot: 100138–201606); mechlorethamine hydrochloride (Shanghai Aladdin Biochemical Technology Co. Ltd., lot: G1414051); doxorubicin (China Food and Drug Control Institute, lot: 130509–201302); Trizol (Coolaber, lot: SL2075); HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China, lot: R123-01); and AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China, lot: Q111-01/02/03).

Genomic DNA and total RNA were isolated using the CTAB and TRIzol methods (Coolaber, Beijing, China), respectively. First-strand cDNA was synthesized using a Reverse Transcription System (Vazyme, Nanjing, China). Sequence BLAST search was performed through NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 28 February 2022) and IWGSC (http://www.wheatgenome.org/, accessed on 28 February 2022) databases. The genomic and cDNA sequences of TaHsfA2e-5D were obtained from the DNA and cDNA of TAM107 by PCR with gene-specific primers. The chromosomal location of TaHsfA2e-5D was determined by PCR using chromosome specific primers with the DNA of CS NT lines as templates. All PCR reactions were performed using high-fidelity Tks Gflex™ DNA Polymerase (TaKaRa, Dalian, China) according to the manufacturer’s recommended procedures. The PCR products were constructed into the pEASY-Blunt cloning vector (TransGen, Beijing, China) for sequencing. Primer sequence information is presented in Table S1. DNAMAN8 software was used for sequence alignment. Cis-acting regulatory elements were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 February 2022). Phylogenetic tree was constructed using MEGA7 software, and sequence logos were analyzed by WebLogo (http://weblogo.berkeley.edu/, accessed on 28 February 2022) platform.