Plant Material
The fresh flower petal during bud stage, middle opening stage, full opening stage and wilting stage (Fig. 1a) and the flower petal, stamen, and pistil at full opening stage and leaves (Fig. 2a) of R. fortunei were collected from Siming Mountain National Forest Park in Ningbo, China. Parts of them were directly used to determinate VOCs, and the remained parts were placed at -80℃ for storage. Wenguang Hu undertook the formal identification of the samples and provide details of specimens deposite in Flora of China. All R. fortunei material was obtained with permission.
Floral scent collection and analysis
The HS-SPME-GC-MS was used to collect the floral scent. Aging the 65µmol/L PDMS/DVB SPME fibers at injection port of GC and set the temperature at 250℃. Weigh 5g of the shredded sample rapidly and put it into an 8mL headspace bottle. And then insert the SPME injector into a sealed bottle. Headspace extraction for 1h at 30℃ water bath with 500 r/min stirring. After the extraction, insert the SPME fibers into injection port of GC and resolve for 5 min immediately. Three biological replicate measurements were performed on each sample.
Chromatographic conditions: Agilent lichrosorb 19091S-433 (30m x 250μm x 0.25μm); the temperature of column is 40℃; the injector was operated splitless at a temperature of 250℃ with He as a carrier gas at 1.0mL/min. The following temperature program was used: initial temperature of 40℃ (2min hold), increase to 160℃ at 3℃/min, 10℃/min ramp to 200℃, followed by a 20℃/min ramp to 300℃ (3min hold), with the port in splitless injection mode 5.
Mass spectrometer conditions: ionization mode: EI; electron energy 70 eV; interface temperature: 250℃; ion source temperature: 230℃; quadrupole temperature 150℃; mass scan range: 15-500 AMU, solvent delay 2.6 min.
Preliminary identification of the VOCs was made by searching the NIST library and checked according to its retention index. The Identification results with a matching degree above 80 (maximum 100) are used [25]. Benzyl benzoate (0.186g/L) was used as an internal standard to quantify the volatile compounds of the floral scent. 200μL of benzyl benzoate was placed in a headspace bottle with the sample for extraction. The content of each VOC(mg/kg)=[Peak area of each VOC/Peak area of internal standard] × the concentration of internal standard × the volume of internal standard (μL) x10-3 / sample weight (kg). Then the aromatic value was calculated for each components respectively according to the threshold of odor [6].
Isolation of the full-length cDNA
Total RNA was isolated from R. fortunei petal using the RNAprep Pure Plant Kit (TIANGEN, China). After passing the NanoDropTM2000 and 1% agarose gel electrophoresis, it was reverse transcribed to first-strand cDNA (CWBIO, China). Download the full length sequence of BAMT gene from plants similar to Rhododendron in GenBank, and then using ClustalW to find their conserved sequence. Finally, a pair of degenerate primers BAMT-F1 and BAMT-R1 were designed for PCR amplification by Primer 5.0 (Table 5). The PCR reaction procedure was determined as follows: predenaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 68°C for 1 min, a total of 35 cycles, and finally, 72°C. Extend for 10 min and store at 4°C (TRANS, China).
The PCR product was detected by 1.0% agarose gel electrophoresis. The fragment was extracted with plastic recycling kit, and then cloned to the vector pEASY-Blunt Zero (TRANS, China) and sequenced by Shanghai Sangon Biotech Company.
Gene-specific primers (5’GSP and 3’GSP, Table 5) were designed based on the sequence of middle segment. The SMARTer® RACE 5’/3’Kit (Takara) was used to isolate the 3' and 5' ends of the cDNA, and spliced a full-length cDNA sequence by DNAMAN.
Bioinformatics analysis
The open reading frame of nucleotide sequence and deduced amino acid sequence was analysed by ORF finder tool on the http://www.NCBI.com website. Conserved domains Research tool was used to predictive gene domain. Molecular weight and isoelectric point of the protein were predicted by the online software ProtParam (http://web.expasy.org/protparam/) on ExPASy. Homology evolution of amino acid sequence and members of the gene family were analysed by DNAMAN software, and constructed phylogenetic tree by MEGA 6.06 software.
Quantitative real-time PCR
To perform quantitative real-time PCR (qRT-PCR) analysis, total RNA was isolated from different flower developing stages and floral parts of R. fortunei. Primers for qRT-PCR were designed based on the RfBAMT cDNA sequence (Table1). EF1α was selected as internal reference gene for each sample (Table 5). qRT-PCR was carried out using the Bio-Rad real-time PCR system with ChamQ™SYBR®Color qPCR Master Mix. The program was: 95℃ degeneration 3 sec, 40 cycles of 95℃ for 10 s, 60℃ for 30s, fluorescent signal acquisition in 60℃. All the qRT-PCR results were presented as means ± SD of three biological replicates. The relative expression level of genes were calculated by the 2 –Δ Δ Ct equation.
Table 5 Primers for experiment.
Primers
|
Sequence(5’→ 3’)
|
Purpose
|
BAMT-F1
|
GTTGTTGAWGTTCTTCACATGAATGG
|
Intermediate fragment
amplification
|
BAMT-R1
|
ACTTCTKCTGGTGATGGTGTRTAYTGAGG
|
5’GSP
|
GCTTGGTGGGCTATTGCTTCCGAT
|
5′-RACE amplification
|
3’GSP
|
GAATTGGTGACGGGTGGTCGCAT
|
3′-RACE amplification
|
UPM
|
TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
|
universal primer
|
CTAATACGACTCACTATAGGGC
|
BAMT-F2
|
AGAGAGAGAGAGATGGAAGTGTATCA
|
Full-length cDNA amplification
|
BAMT-R2
|
ATAAAACATCACAACAATACCAACAT
|
BAMT-F
|
TCTACTGTCCCTTGGAGTGCCT
|
Primer for qRT-PCR
|
BAMT-R
|
TCATACCTCTGGAAAACCTTGTCTA
|
EF1α-F
|
TGTCATCGATGCTCCTGGAC
|
Reference Primer
|
EF1α-R
|
TCTCGGGTCTGACCATCCTT
|