Rnaprep pure kit

The cotton (86 III 72 glanded and 86 III 72 glandless) ovules of 30 DPA and young leaves were collected, and total RNA were extracted using an RNAprep Pure Kit (Tiangen Biochemical Technology (Beijing, China) Co., Ltd.). Each sample included three individual biological replicates. cDNA was synthesized using HiScript^®Q RT SuperMix for qPCR (Vazyme, China) according to the protocol for qRT-PCR analysis. The qRT-PCR primers of genes were designed through Primer Premier 5.0 (Table S11). The qRT-PCR was carried out in a 20 uL volume, with three replicates for each sample including 10 μL 2 × SuperReal Color PreMix, each of the forward and reverse primers 0.6 μL (10 μM), 1 μL cDNA (50 ng μL^–1), 0.4 μL 50 × ROX reference dye, and 7.4 μL RNase-free ddH₂O. The qRT-PCR reaction conditions were 95 °C for 15 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s in the PCR stage. Data acquisition and analyses were performed using the QuantStudio^TM Real-Time PCR Software (Thermo Fisher Scientific, Waltham, MA, USA). The data were normalized by the internal control gene UBQ7 (DQ116441) and the relative expression level was calculated using the 2^–ΔΔCT analysis method [36 (link)].

Total RNA was extracted with an RNAprep Pure kit (Tiangen, China), which was suitable for plants rich in polyphenols and amylase. The quality and concentration of the RNA were confirmed by 1% agarose gel electrophoresis and a spectrophotometer, and DNaseI was used to remove the genomic DNA. cDNA synthesis was performed by strictly following the manufacturer’s protocol for ReverTra Ace qPCR RT Master Mix (TOYOBO, Japan). Total genomic DNA was extracted from the cotton leaf tissues by the CTAB method (Song et al., 1998 ).

Total RNA was extracted from rice leaves using RNAprep Pure Kit (TIANGEN, China). The quality and quantity of the purified RNA were assessed with a NanoDrop-1000 (NanoDrop, USA). First-strand cDNA was synthesized using ReverTra Ace (Toyobo, Japan). Primers were designed to cover the complete open reading frame of each GLO gene (Additional file 8). To construct the vectors for protein expression in yeast, a 6 × His-tag was fused to the N-terminus of GLO1, GLO3 and GLO4 [23 (link)], and then these modified sequences (NHisGLO1, NHisGLO3 and NHisGLO4) were cloned into pYES3 and pYES2 vectors. To generate GLO-overexpression transgenic lines, each GLO sequence was cloned into pYLox.5 vector. To generate GLO-silencing transgenic lines, primers were designed to amplify the interfering fragment to guarantee the specificity of the silencing (Additional file 8), each specific fragment was then cloned into the RNAi vector pYLRNAi.5. To generate CRISPR-Cas9 knockout lines, specific targeting sequences were synthesized and cloned into pYLCRISPR/Cas9P_ubi vector (Additional file 4) [46 (link)]. (pYLox.5, pYLRNAi.5 and pYLCRISPR/Cas9 vectors were kindly provided by Dr. Yao-Guang Liu, College of Life Sciences, South China Agricultural University).

A total of 12 samples [4 treatments (control, ZnSO₄, ZnMet, and ZnGly) × 3 biological replications] were used for transcriptome analysis. Total RNA was extracted using a TIANGEN RNAprep Pure kit (TIANGEN, catalog # DP441), according to the manufacturer's instructions. RNA concentrations were measured using a Qubit^® RNA Assay Kit in a Qubit^® 2.0 Fluorimeter (Life Technologies, CA, USA). The assessment of the RNA integrity number (RIN) was performed using an RNA Nano 6000 Assay Kit and the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Genomic DNA was prepared by Bust n’ Grab method [34 (link)]. Total RNA of each sample was prepared using an RNAprep pure kit (Tiangen Biotech, Co., Ltd, Beijing, China) following the manufacturer’s recommendations. RNA samples were stored at −80°C until used. 2 μg of each total RNA was subjected to reverse transcription using the Fast Quant RT Kit (Tiangen Biotech, Co., Ltd, Beijing, China).

Total RNA was extracted using an RNA prep pure kit (TIANGEN Biotech, Beijing, China, http://www.tiangen.com/en/ (accessed on 21 May 2023)). A 1 μg portion of total RNA was reverse-transcribed by priming with oligo (dT18) in a 20 μg reaction based on the Prime Script Reverse Transcriptase kit (TaKaRa, http://www.takara-bio.com (accessed on 20 March 2023)). The value of β-actin and α-tubulin mRNA were used as the internal control [66 (link)]. The primers in this study were listed in Table S13.