Hipure hp plant rna mini kit

RNA was extracted from the above-mentioned triticale leaves using Hipure HP Plant RNA Mini Kits (Magen). Using these RNA as templates, we performed reverse transcriptase PCR (RT-PCR) to synthesize cDNA sequences using SMARTScribe genome Reverse Transcriptase (TaKaRa, Dalian, China). Synthesized cDNA was stored at −20°C before being sequenced. Based on RNA-Seq sequencing results, the sequence of Unigene c51971 (708 bp) was studied in this research, and specific primers for 5′- and 3′-RACE using SMARTer RACE kits (Supplementary Table S1) were designed. The RACE procedure was as follows: 94°C for 2 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 72°C for 35 min, and 16°C for 10 min. The PCR products were recovered and purified using 1.0% agarose gel electrophoresis. The purified PCR products were ligated to the pMD19-T vector (TaKaRa, Dalian, China) and transformed into TOPO10 cells (TIANGEN, China). Positive clones were identified and sequenced. The open reading frame (ORF) of the full-length cDNA sequence was obtained by splicing the sequencing results using an online tool CAP3¹ (Shang et al., 2019 (link)) and the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI-BLAST). Sequence alignment analysis preliminarily identified the sequence as a triticale NAC gene, which was preliminarily designated TwNAC01.

To obtain transgenic Arabidopsis plants, the coding sequence containing the stop codon of TwNAC01 was amplified by RT-PCR and cloned into the KpnI and XbaI restriction sites of the pCAMBIA1300-35S vector (TaKaRa, Dalian, China) under the control of the 35S promoter of the cauliflower mosaic virus (CMV). The primers containing the KpnI and XbaI restriction sites are listed in Supplementary Table S1. The recombinant vector pCAMBIA1300-35S-TwNAC01 and the empty vector pCAMBIA1300-35S-VC were introduced into A. tumefaciens strain GV3101 (TaKaRa, Dalian, China). Finally, transgenic Arabidopsis plants were generated using the A. tumefaciens-mediated floral dipping method (Clough and Bent, 1998 (link)). To generate homozygous progeny, T1 and T2 seeds were selected on kanamycin (50 mg l⁻¹) plates. T3 transgenic and WT plants of A. thaliana were watered once at the rosette stage and then subjected to drought stress for 25 days. RNA was then extracted from the leaves and roots of both transgenic and WT A. thaliana using Hipure HP Plant RNA Mini Kits (Magen, China). TwNAC01 gene expression levels were then measured using semi-quantitative analysis with gene-specific primers (Supplementary Table S1) following the same qPCR protocol. Representative lines overexpressing TwNAC01 were used for further analysis.

The total RNA was extracted from samples treated with NaCl using HiPure HP Plant RNA Mini Kit (Magen, shanghai, China). Then, the RNA was used for cDNA synthesis with the PrimeScript™ RT reagent kit with gDNA Eraser (Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was performed according to the supplier’s instructions of the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China) in the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR reaction system consists of one 5 µL cDNA sample, 0.6 µL primers, and 7.5 µL SYBR Green (Accurate Biotechnology, Hunan, China), 1.9 µL nuclease-free H₂O, and the reaction volume was 15 µL. The PCR reaction was performed with the following conditions: 30 s at 95 °C, 40 cycles of 5 s at 95 °C, and 30 s at 60 °C. The grapevine β-actin (XM_034827164.1) were used as the internal references. All experiments were repeated at least three times and all the primers used in this study were listed in Table S1.