Plant Materials and Growth Conditions
Camellia nitidissima tissues were collected from the National Camellia Germplasm Resource Bank (Guangxi, China, E 108°20'53", N 22°49'11", 75m above sea level) in Nanning, Guangxi Province, China. The materials were frozen in liquid nitrogen and stored at -80℃ for later use.
Nicotiana benthamiana was used in the transient transformation and stable transformation experiments. The seedlings were grown in a growth chamber (RDN-1000E, Ningbo Yang hui Instrument Co. Ltd, China; temperature: 25℃, humidity: 76%, illumination: 6000Lx, light cycle: 16/8h).
Cloning of CnF3'H
Total RNA was isolated using an RNAprep Pure Extraction Kit (DP441, Tiangen Biochemical Technology, China) and RNA integrity was determined based on 1.5% agarose gel electrophoresis analysis. A PrimeScript Ⅱ 1st Strand cDNA Synthesis Kit (6210, TaKaRa, Japan) was used to synthesize the cDNA for gene cloning experiments, according to the manufacturer’s instructions. We designed a pair of specific primers (S1.1) using Primer 3 (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) according to the transcriptome data. The PCR products were cloned into a T-vector (CT501, TransGen Biotech Co., Ltd, China) for sequencing. The full-length CnF3'H gene was assembled and verified based on sequence analysis.
Sequence Alignment and Phylogenetic Analysis
BioEdit and NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to align the sequences (McGinnis and Madden 2004). NCBI ORFfinder (http://www.ncbi.nlm.nih.gov/projects/gorf/) was used to identify the reading frame (Wheeler et al., 2006), and ProtParam (https://web.expasy.org/protparam/) was used to analyze the protein molecular weight and isoelectric point (Wilkins et al. 1999). Amino acid sequence alignment was performed by the software of DNAMAN and the phylogenetic tree was constructed with MEGA 5.0 software, using the neighbor-joining (NJ) method and 1000 bootstrap replicates (Thompson et al. 1994).
Quantitative PCR Analysis of CnF3'H
We chose GAPDH as the reference gene (S1.2) and a pair of unique primers of F3’H (S1.3) for quantitative PCR analysis. A PrimeScript RT reagent Kit with gDNA Eraser (RR047, TaKaRa) was used to synthesize the first-strand cDNA. The quantitative PCR reaction system was constructed using SYBR Prime Ex Tap Ⅱ (Tli RNaseH Plus) (RR420, TaKaRa). The reaction was performed on a QuantStudio® 7 Flex (Applied Biosystems, USA), and the reaction procedure was as follows: pre-denaturation at 95℃ for 30 s; 98℃ 5 s, 60℃ 34 s, 40 cycles; 95℃ 15 s, 60℃ 1 min, 95℃ 15 s. The relative expression quantity of CnF3'H was measured in different tissues and in different development periods by the 2 (- Δ Δ CT) method (Livak and Schmittgen 2001).
High-Performance Liquid Chromatography Analysis
An NF555 colorimeter (Nippon Denshoku Industries Co., Ltd, Japan) was used to detect the color indicator of the petals. HPLC analysis was used to assess the flavonoid, polyphenol and anthocyanin constituents. We first ground fresh sample (0.6 g) in liquid nitrogen, then added 5 mL of extraction solution (methanol: water: formic acid: trifluoroacetic acid = 70: 27: 2: 1). The samples were extracted in the dark for 24 hours with occasional shaking. After extraction, the samples were filtered with absorbent cotton to remove residues and passed through an organic microporous filter membrane (0.22 cm) (ANPEL Laboratory Technologies Inc., (Shanghai, China). The filtrate underwent HPLC analysis.
An Agilent Technologies 1260 Infinity (Agilent Technologies, Inc., Germany) equipped with Waters SunFire C18 column (4.6×250 mm, 5μm) (Waters Co., USA) was used for HPLC. The column temperature was 30℃., the flow rate was 1.0 mL/min; and the injection volume was 10 μL. The elution mobile phases were A: 2% formic acid solution and B: pure acetonitrile. The elution procedure for flavonoids was as follows: 0-5 min, 20% B; 5-15 min, 20% up to 40% B; 15-20 min, 40% up to 60% B; 20-20.2 min, 60% down to 20% B; 20.2-24 min, 20% B. The detection wavelength of flavonoids was 350 nm. The elution procedure for polyphenols was as follows: 0-9 min, 98% down to 90.7% B; 9-15 min, 90.7% B; 15-20.5 min, 90.7% down to 85% B; 20.5-29.5 min, 85% down to 75% B; 29.5-30 min, 75% up to 98% B; 30-34 min, 98% B. The detection wavelength of polyphenols was 278 nm.
Subcellular Localization of CnF3'H Protein
We designed a pair of primers (S1.4) according to the EXclone Kit instructions (exv09, Hangzhou Biogle Co. Ltd, China) for the vector construction. The overexpression vector was transformed to Agrobacterium tumefaciensGV3101 strain via the thermal shock method (Gelvin 2003). To perform tobacco infiltration analysis, the transformed agrobacteria were suspended in induction medium (10 mM/L MES+10 mM/L MgCl2+100 uM/L acetosyringone) and injected into the Nicotiana benthamiana leaf (Ma et al. 2012). We then detected the GFP signal 2~5 days after injection using LSM510 Meta confocal microscope (Zeiss, Germany) (Yamaguchi et al. 2005).
Tobacco Transformation Analysis of CnF3'H
To verify the functionality of CnF3'H, we heterologously transformed Nicotiana benthamiana by the leaf plate method. PCR primers (S1.5) were applied using T5 Direct PCR Kit (Plant) (TSE011, TSING KE Biological Technology, China) to positively identify rooting plants. The PCR procedure was as follows: pre-denaturation at 98℃ for 3 min, denaturation at 98℃ for 10 s, annealing at 65℃ for 10 s, extension at 72℃ for 1 min and 30 s, 30 cycles, extended at 72℃ for 5 min. The PCR products were analyzed by 1% agarose gel electrophoresis. After the positive plants had flowered, we collected the flowers, froze them in liquid nitrogen and stored the samples at -80℃. Quantitative PCR was used to measure the relative expression of CnF3'H with the reference gene of 18S (S1.6). The total flavonoid, total polyphenol and total anthocyanin contents in the flowers were measured by a spectrophotometric assay (Jiang et al. 2019).Individual flavonoids, anthocyanins and polyphenols in the flowers were also determined by HPLC and compared with the control group.