The peptides GMA4CG_WT and GMA4CG_V1–V6 were chemically synthesized with an initial purity of about 80%–85% from multiple commercial vendors and further purified using a C‐18 reverse phase HPLC as previously described (Velivelli et al., 2020 (link)). The HPLC fractions containing the peptide were lyophilized and resuspended in nuclease‐free water. Peptide concentrations were determined using the BCA assay. For peptides containing Trp, the peptide concentration was also determined using NanoDrop 2000c spectrophotometry (Thermo Fisher Scientific) to confirm concentrations determined by the BCA assay.
Nanodrop 2000c spectrophotometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 2000c is a compact, UV-Vis spectrophotometer designed for the measurement of nucleic acids and proteins. It utilizes a patented sample retention system that requires only 1-2 μL of sample to perform high-accuracy absorbance measurements across a wavelength range of 220-840 nm. The NanoDrop 2000c provides fast and reliable results for a variety of life science applications.
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5 protocols using nanodrop 2000c spectrophotometry
1
Peptide Synthesis and Purification
2
Extracting DNA from Ornamental Plant Cultivars
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Fresh leaves of B. spectabilis and 53 cultivars were collected from the Germplasm Resource Nursery of Ornamental Plants (Table S1 ), Guangzhou Institute of Forestry and Landscape Architecture, Guangzhou, China. The leaf tissues from an individual plant were sampled for all 53 cultivars. The leaves were kept in aluminum ziplock bags and transported back to the laboratory to be kept at −80 °C prior to DNA extraction. Total DNA extraction was carried out using DN15 Plant DNA Mini Kits (Aidlab Biotechnologies, Beijing, China) according to the manufacturer’s protocol. DNA quantification and quality were estimated through Nanodrop 2000 C spectrophotometry (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% (w/v) agarose gel.
3
Quantitative Analysis of EC and SE Gene Expression
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The expression of EC and SE related DEGs was detected by qRT-PCR (Bio-Rad CFX96, USA). RNA was extracted using a polysaccharide polyphenol total RNA kit (Tiangen, Beijing, China). Ten µl of RNA was extracted from each sample. The quality and quantity of RNA were assessed by 1.2% agarose electrophoresis and NanoDrop 2000c spectrophotometry (Thermo Scientific, USA) respectively. The reverse transcribed with AG Evo M-MLV RT Mix Reverse Transcription Kit, removes DNA residues from the genome with gDNA Clean Reaction Mix Ver.2 and Fluorescence quantitative PCR was using the SYBR® Green Pro Taq HS Master Mixed qRT-PCR Kit. The reaction was comprised of 1 µl of cDNA template, 10 µl of 2 × SYBR Green Pro Taq HS premix, 0.4 µl each of primer F and primer R (10 µmol·L-1), and RNase-free H2O to 20 µl. The cycling conditions were as follows: pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and renaturation at 60 °C for 30 s. Each sample was tested 3 times. Normalization was performed using the EF1-α gene as the internal control [23 (link)]. Data were analyzed by Microsoft Excel 2019 software, and the relative expression was calculated by the 2-ΔΔCt method [24 (link)]. The primers were designed using Primer-BLAST, and they are listed in Table S1 .
4
Genomic DNA Extraction and Sequencing Library Preparation
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Total DNA was extracted from silica-dried leaf material using a modified extraction method described by Yang et al. [82 (link)]. The quality and concentration of the extracted DNA were detected by 1.0% agarose gel electrophoresis and by a NanoDrop 2000C spectrophotometry (Thermo Fisher, US). The extraction genomic DNA (approximately 1 μg) was subjected to random degradation by Covaris (E210), and then fragments with a size of 200–400 bp were selected by using Agencourt AMPure XP-Medium kit. The selected fragments were amplified after suffering from end repair, 3′-adenylation and adaptor ligation. Then, they were heat denatured to single strand after purification. The single strands were circularized, and single strand circle DNA was obtained as the final library. The final library was sequenced by BGISEQ-500 (BGI, Shenzhen, China) to generate raw reads. The details of the quantity and quality of raw reads, and coverage depth of the assembled genomes were provided in Additional file 2 : Table S4.
5
DNA Extraction and ITS2 Amplification
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The total DNA extractions were performed utilizing the Plant Genomic DNA Kit (Tiangen Biotech (Beijing) Co. Ltd., China) according to the manufacturer’s instructions. The DNA concentration and quality were assessed by Nanodrop 2000C spectrophotometry (Thermo Fisher Scientific Inc., China) and 0.8% (w/v) agarose gel electrophoresis at 120 V for 40 min (Bio Rad Laboratories Inc., USA), respectively. According to the procedure provided by Chen et al.31 (link), PCR amplification of ITS2 was performed in 50 μL reaction mixtures using 2 × Taq MasterMix (AidLab Biotechnologies Co. Ltd., China), with the annealing temperature increased to 58 °C for 40 cycles. The PCR products were purified by Universal DNA Purification Kit (Tiangen Biotech (Beijing) Co. Ltd., China) and were quantified and assessed by Nanodrop 2000C spectrophotometry and 2% (w/v) agarose gel electrophoresis at 120 V for 30 min. Resulting DNA products were used in the following assay.
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