Dnaman program

The transcript levels of TaOSCA1.4 in Fielder organs were analyzed via qRT−PCR. The primers used were as follows:
The first-strand cDNAs were synthesized from 2 µg of RNA per sample using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). The qRT−PCR aplifications were performed as described by Zhao et al. (2020) (link). Amplification of TaActin was used as an internal control for data normalization. The experiments were independently replicated three times under identical conditions. The complete alignment of multiple coding sequences and translations of nucleotides into amino acid sequences were performed using the DNAMAN program (version 5.2.2; Lynnon Biosoft, Canada). The prediction of the transmembrane structure was performed using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM). Collinearity analysis was performed using the Triticeae-Gene Tribe (TGT, http://wheat.cau.edu.cn/TGT/).

Cells (1×10⁵ per well in a 6-well plate) were cultured and treated as described above. Total RNA was isolated using an RNApure Tissue Kit (CWBiotech; Beijing, China) according to the manufacturer’s instructions. Primers were designed using the DNAMAN program (V. 6.0.3; Lynnon Biosoft, Canada). First-strand cDNA was synthesized from total RNA using ReverTra Ace-α (Toyobo; Osaka, Japan). Quantitative real-time PCR was performed by LightCycler-based SYBR Green I dye detection with UltraSYBR Mixture (CWBiotech). Gene expression was quantified by the 2^-ΔΔCT method [35 (link)].

Fasting venous blood was collected, and peripheral leukocytes were isolated with the Human Leukocyte Isolation system (Haoyang Biological Products Technology Co., Ltd., Tianjin, China). Genomic DNAs were extracted with the QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, United States). The promoter region of the human TFEB gene were generated with PCR and directly sequenced. Two overlapped DNA fragments, 705 bp (−1312 ∼−608 bp) and 801 bp (−657 bp ∼ +144 bp), were overlapped, covering the TFEB gene promoter region. The PCR primers were designed using the human TFEB genomic sequence (National Center for Biotechnology Information GenBank accession no. NC_000006.12). PCR products were directly and bi-directionally sequenced on a 3500XL genetic analyzer (Thermo Fisher Scientific, Inc., Waltham, MA, United States) by Sangon Biotech Co., Ltd. (Shanghai, China). DNA sequences were then compared with the wild-type TFEB gene promoter using the DNAMAN program (Version 5.2.2, Lynnon BioSoft, Quebec, Canada), and regulatory variants including single-nucleotide polymorphisms (SNPs) were identified. Wild and variant TFEB gene promoters were analyzed using TRANSFAC and JASPAR programs to predict the binding sites for the transcription factor affected by regulatory variants.