5.1 Plant Materials and Reagents
Plants of D. officinale were grown at 25℃ during day and 23℃ at night with 60–70% relative humidity and a light/dark cycle of 14/10 h in the herbal garden of Anhui University of Chinese Medicine, Hefei, China.
Standard products of (+)-Glucose, Dendrobine, Rutin and Naringenin (> 98% purity) were bought from Chengdu Push Bio-technology CO., Ltd. Methanol (HPLC grade) were purchased from Oceanpak, and CTAB-PBIOZOL reagent used for total RNA extraction was purchased from Bioflux (Beijing, China). HPLC-grade water was prepared by water purification system from Pall Filter Co., Ltd. (Beijing, China). Plant growth regulators (PGRs) include 6-BA (6-Benzylaminopurine), α-NAA (α-Naphthylacetic acid) and 2, 4-D (2, 4-dichlorophenoxyacetic acid) were purchased from Solarbio. Agents for PLBs induction and propagation were bought from Sinopharm group and all of the other agents were analytical grade.
5.2 Protocorms and PLBs of D. officinale Induction and Propagation
Capsules approaching mature, leaves, stem tips, stem fragments, and stems with nodes of D. officinale were collected, and sterilized to induce protocorms and PLBs (Capsules were punctured to release seeds as explants on medium under sterile condition), each explant was performed more than 30 duplicates. They were inoculated in 1/2 MS medium + 2, 4-D 0.5 mg·L− 1 + 30 g·L− 1 sucrose + 7 g·L− 1 agar at pH 5.6 to 5.8, and placed in the tissue culture rooms at 70% relative humidity, 1600 lx illumination and a light/dark of 16/8 h for 60 d, to screen the optimal explants for protocorms and PLBs induction.
Protocorms and PLBs induced above were collected as explants to be cut into pieces of about 0.5 cm × 0.5 cm to proliferate much more PBLs, and the optimum culture protocol for the PLBs of D. officinale proliferation was screened. An L16(45) orthogonal experiment without regarding the interactions among factors was performed, the factors and their levels were selected based on our previous experiments and the reports on literatures [51]. Four different gradients of MS medium such as 1/4MS, 1/2MS, MS and 2MS were used as test media (Factor A), the plant growth regulators (PGRs) combinations with different concentration of α-NAA (Factor B) with concentrations of 0.1 mg·L− 1, 0.2 mg·L− 1, 0.5 mg·L− 1 and 1.0 mg·L− 1, 6-BA (Factor C) with 0.5 mg·L− 1, 1.0 mg·L− 1, 1.5 mg·L− 1 and 2.0 mg·L− 1 and 2,4-D (Factor D) with 1.5 mg·L− 1, 2.0 mg·L− 1, 2.5 mg·L− 1 and 3.0 mg·L− 1 were arranged to screen the optimal PGRs combination. It was found that the materials in tissue culture with natural additives and fruit juice grow much better than those without natural additives and fruit juice [52, 53], in the PLBs proliferation processing potato juice was selected as additive to accelerate PLB growth and propagation. 200 g fresh potato was chopped into small pieces, added was mixed with purified water to 1 L and boiled to a mush. The mushy potato was passed through the gauze to collect the juice as additive. The concentration gradients of potato juice (Factor E) were 50 g·L− 1, 100 g·L− 1, 150 g·L− 1 and 200 g·L− 1. All of the full test media with pH 5.6 ~ 5.8 for PLBs proliferation were included basic medium, PRGs combination, potato juice, sucrose (30 g·L− 1) and agar (7 g·L− 1), and the design of orthogonal experiment table head was showed in Additional file 11. Each group has 10 bottles with 4 pieces, and the culture condition was light/dark for 16/8 h per day, the illuminance and the culture temperature were at 1600 lx and (25 ± 1) °C. Observations and statistics were carried out to analyze the propagation coefficient two months later since inoculation. The formula of propagation coefficient was calculated as follow: Propagation coefficient (%) = (FW1 – FW0)/ FW0 × 100%, FW0 and FW1 mean the fresh weight before inoculation and after culture, respectively.
5.3 Ultrasound-Assisted Extraction (UAE) or Hot Water Extraction (HWE) of Total Polysaccharides
Stems, leaves, roots and the whole plants from about 3 years old D. officinale and PLBs were harvested, cleaned, and dried in an oven at 50 ℃ to a constant weight. Samples were ground to a fine powder using the pulverizer and sieve the powder through a 40 meshes sieve. The water-soluble polysaccharides in D. officinale were extracted by UAE method [41] and HWE method [8] with a few modification and finally better one was selected for the extraction of polysaccharides in D officinale from these two methods.
The modified UAE method was as follows: 10 mL of double distilled water was added into each 0.2 g pulverous sample, and the samples were homogenized in the ultrasonic cleaner at 40 ℃ ,40 Hz for 0.5 h, then filtered through filter-paper and the filtrate was obtained. Repeat above steps one more time and collect all filtrate. The filtrate was concentrated at 55℃ until its volume was down to 10 mL. 40 mL of ethanol was added into the concentrated filtrate, centrifuged at 4,000 rpm for 5 min, and discarded the supernatant. The samples were dissolved in 25 mL of pure water. 25 mL of savage reagent was added to remove impurities such as protein and nucleic acid, centrifuged at 1000 rpm for 5 min. 20 mL of the supernatant was transferred into a 50 mL volumetric flask and mixed with pure water to a constant volume of 50 mL for polysaccharides extraction use. The method HWE with a few modifications was as follows: firstly, 0.3 g samples were placed in a round-bottomed flask, and add 200 mL of water. Then the sample solution was heated and refluxed for 3 h. Let the reflux extract cool off to room temperature before filtering it. The filtrate was transferred into a 250 mL volumetric flask, and mixed with double distilled water to a constant volume of 250 mL. 2 mL of sample solution was taken into a 15 mL centrifugal tube and 10 mL of solute ethanol was added, shake it and refrigerate it for 1 h followed by centrifugation at 1000 rpm for 20 min. Discard the supernatant and wash the precipitate twice with 8 mL of 80% ethanol. The final precipitate was dissolved in heated water and transferred to a 25 mL volumetric flask. Let the solution cool off before mixing it with pure water to a constant volume of 25 mL for polysaccharides extraction use. Thus, the polysaccharide extracts by the two methods have been prepared, respectively.
5.4 Determination of Total Polysaccharides
1 mL of each polysaccharide extract was transferred into a 10 mL test tube, then add 1 mL of 5% phenol and vortex quickly. The solution was mixed thoroughly and 5 mL of concentrated sulfuric acid was added, shaken, and placed in a water bath at 100℃ for 20 min. Then the solution was placed in ice bath to cool for 5 min. The absorbance of the sample solution was measured at wavelength of 488 nm using ultraviolet visible spectrophotometer with 1 mL of water as a blank and the parallel detection was conducted three times. The standard curve was prepared from the D-glucose reference. Each sample was assayed in three times and the content showed as the weight of polysaccharides to the dried weight of materials.
5.5 Extraction and Determination of Total Alkaloids
The sample powder (0.5 g) was put into a 125 mL ground-mouth flask and mixed with 30 mL of petroleum ether. The sample mixture was placed in a constant temperature water bath (35℃) to degrease for 30 min, then remove the supernatant and evaporate the petroleum ether. The pH value was adjusted with a proper amount of ammonium hydroxide and add 10.0 mL of chloroform. Then the solution was refluxed for 2 h in a water bath at 80 ℃. Let it cool for 20 min in room temperature before filtering. The filtrate was obtained as total alkaloids solution.
5 mL of total alkaloids solution was mixed with 5 mL of chloroform, then 5.0 mL of pH = 4.5 buffer and 2.0 mL of 0.04% bromocresol green solution were added sequentially. Shake the mixture vigorously for 3 min and let it stand for 30 min before filtering it. And 5.0 mL of filtrate was mixed with 1.0 mL of 0.01 M NaOH-ethanol solution and vortex it. The absorbance of the samples was measured at 620 nm using UV-visible spectrophotometer with chloroform as blank. The standard curve was prepared from the Dendrobine as reference (> 98% purity) bought from Chengdu Push Bio-technology CO., Ltd. (0.857, 1.714, 2.571, 3.429, 4.286, and 5.143 µg·mL− 1). The parallel detection was carried out three times and the content showed as the weight of total alkaloids to the dried weight of materials.
5.6 Extraction and Determination of Total Flavonoids
The samples powder (1.00 g) was dissolved in 50 mL of 70% ethanol and refluxed at 60℃ for 2 h, then filtered. The filtrate was mixed with 70% ethanol again to a constant volume of 50 mL as total flavonoids extracts.
1 mL of total flavonoids extract was mixed with 5 mL of 70% ethanol. Then add 1 mL of 5% NaNO2 and mix it well. Stand for 6 min and add 1 mL of 10% Al(NO3)3 and mixi it well. Let it stand for 6 min before adding 10 mL of 1 M NaOH and mix it with 70% ethanol to 25 mL, and stand for 15 min. The absorbance of the sample solution was measured at 510 nm using ultraviolet visible spectrophotometer. The standard curve was prepared from Rutin as reference (2.208, 4.416, 6.624, 8.832, and 11.040 µg·mL− 1). Each sample was assayed in three times and the content showed as the weight of total flavonoids to the dried weight of materials.
5.7 Extraction and Quantification of Naringenin
Naringenin was extracted by the optimized methods: Solvent, methanol (20 mL) mixed with 20% hydrochloric acid (5 mL); Particle size of dry powder of D. officinale < 0.355 mm; temperature, 80℃; Condensation reflux extraction time, 90 min; 20 mL of supernatant was evaporated using the rotating evaporator until it was dry. 5 mL of methanol was added to dissolve naringenin and the solution was filtered through a 0.22 mm Nylon membrane filter before HPLC analysis.
HPLC analysis was performed on an Agilent HPLC system, including quaternary solvent management, sampler manager, separation system, detection systems. An Agilent C18 column (4.6 mm × 250 mm, 5 µm) was used at a column temperature of 30℃. The standards and samples were separated using a gradient mobile phase consisting of 0.2% phosphate buffer (A) and methanol (B). The gradient elution program was as follows: 0.0–5.0 min, 0–25% B; 5.0–10.0 min, 25–30% B; 10.0–25.0 min, 30–40% B; 25.0–45.0 min, 40–55% B; 45.0–50.0 min, 55–60% B. The flow rate was 1 mL·min− 1, and the naringenin was detected at 280 nm. The injection volume was 10 µL.
Naringenin stock solution was prepared and diluted to an appropriate concentration for the preparation of calibration curve. The calibration curve was prepared according to the linear plots of the naringenin concentration versus the corresponding chromato-graphic peak area.
5.8 Total RNA Extraction, Construction of cDNA Libraries and RNA-Seq
Ethanol precipitation protocol and CTAB-PBIOZOL reagent was used to purify total RNA from PLBs and leaves according to the manual instructions, and each sample has three biological duplicates. 80 mg tissue samples were ground into powder in liquid nitrogen and the powdered samples were transferred into 1.5 mL of preheated 65℃ CTAB-pBIOZOL reagents. The sample solution was incubated by thermostatic mixer at 65 °C for 15 min to completely dissociate the nucleoprotein complexes. The supernatant was obtained by centrifuging the solution at 12000 × g, at 4℃ for 5 min. The supernatant was mixed with 400 uL of chloroform (per 1.5 mL of added CTAB-pBIOZOL reagent), and the sample mixture was centrifuged at 12000 × g, at 4˚C for 10 min. The supernatant was transferred to a new 2 mL tube and added 700 µL of acidic phenol and 200 µL of chloroform into it followed by centrifugation at 12000 × g, at 4˚C for 10 min. The aqueous supernatant was collected and an equal volume of chloroform was added into it followed by centrifugation at 12000 × g, at 4℃ for 10 min. An equal volume of isopropyl alcohol was added into the supernatant and the mixture was placed at -20℃ for 2 h to precipitate before centrifuging it at 12000 × g, at 4℃ for 20 min, and the supernatant was removed. The RNA pellet was washed with 1 mL of 75% ethanol before being air-dried in a biosafety cabinet and dissolved in 50 uL of DEPC-treated water. Subsequently, qualification and quantification of total RNA were performed using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purify mRNA. The purified mRNA was split up into small pieces with fragment buffer at an appropriate temperature. The random hexamer-primed reverse transcription was performed to synthesize the first-strand cDNA followed by the second-strand cDNA synthesis. Then, A-Tail Mix and RNA Index Adapter were added to end the repair. The cDNA fragments obtained from above mentioned steps were amplified by PCR. The products were purified by Ampure XP Beads and dissolved in EB solution. The product was verified for quality control on an Agilent 2100 bioanalyzer. The double-stranded PCR products were denatured and cycled by the splint oligo-nucleotide sequence to obtain the final library, and the single-stranded circular DNA (ssCir DNA) was formatted into the final library. The established library was amplified with phi29 to obtain DNA nanoball (DNB), each molecular of which had over 300 copies. The DNBs were added into the patterned nanoarray and 100 base pairs of reads generated on the BGIseq500 platform.
5.9 Functional Annotation of Unigenes by Reference Genome
The original sequencing data (original polymerase reads) produced from raw data by Pacific Bioscience Sequel were processed by the SMRT analysis package version 2.3.0 according to the IsoSeq protocol (Pacific Biosciences, https://www.pacb.com/products-and-services/analytical-software/smrt-analysis). ROIs (reads of insert) were generated by original Polymerase reads, which had full passes > 0 and the predicted consensus accuracy > 0.75. According to whether the primers are 5'primers, 3'primers or poly-A tails, ROIs with a minimum length of 300 bp were divided into non-full-length and full-length transcribed sequences. The full-length sequences were processed to De novo consensus isoforms by ICE (Iterative Clustering for Error Correction) algorithm and then polished by Quvier quality-aware algorithm. The De novo consensus isoforms of high quality (the expected Quiver accuracy ≥ 0.95) from each library were combined and then rid redundancy by using CD-HIT [54] founded on the sequence similarity to get final unique full-length isoforms.
The final complete subtype was mapped to SwissProt (manually annotated and reviewed protein sequence database), NR (NCBI non-redundant protein sequence), KEGG (Kyoto Encyclopedia of Genes and Genomes), NT (NCBI non-redundant nucleotide sequence) and the KOG (Clusters of Eukaryotic Orthologous Groups) database through Blast software (version 2.2.23) [55] with default parameters (under a threshold E-value ≤ 10–5) to obtain the isoform annotations. GO (Gene Ontology) annotations and functional classifications were acquired using Blast2GO program (version 2.5.0, E-value ≤ 10–5) [56] according to NR annotations. InterProScan5 software (version 5.11-51.0) [57] was used to acquire annotations from InterPro database.
5.10 Identification of Differentially Expressed Genes (DEGs)
All clean reads were mapped to the reference full-length transcriptome using BLAST software. Gene expression levels were determined by the number of full-length transcriptions, which belong to a cluster after ice clustering and CD-HIT process. Then use the total isoforms’ counts to normalize the counts in each sample. Differentially expressed genes (DEGs) were acquired using DEseq2 with Q value (adjust P value) < 0.001 and fold change (FC) ≥ 2 or ≤ -2 [58]. These DEGs were then carried into GO and KEGG enrichment with Phyper in R package using Q value ≤ 0.05 as default.
5.11 Comparative Analysis of Putative Genes Expression in Metabolites Biosynthesis Pathway
The amino acid sequences of putative genes involved in polysaccharides, alkaloids and flavonoids biosynthetic pathway were searched in National Center for Biotechnology Information (NCBI) database (Additional file 12). All amino acid sequences of putative genes were mapped to the protein information from RNA-Seq using BLAST software to get the average FPKM value of each genes in each sample. Heatmaps of putative genes involving in polysaccharides, alkaloids and flavonoids biosynthesis were drawn based on FPKM value.
Statistical analysis
The experimental data are expressed as the mean ± standard deviation of three independent biological replicates. The statistical differences between samples were analyzed using two-way analysis of variance by SPSS (version 17.0). Values at P < 0.01 were considered statistically significant.