Experimental materials
The sterile seedlings of S. miltiorrhiza preserved in this experiment were subcultured by MS medium every 45 days. S. miltiorrhiza hairy roots induced by Agrobacterium rhizogenes (ATCC15834 strain) preserved in our laboratory were subcultured every 30 days. Nicotiana benthamiana was cultured at 25℃ for 12 h under light conditions. head, stem, leaf, flower and root of S. miltiorrhiza were collected from two-year-old S. miltiorrhiza, frozen in liquid nitrogen and stored at -80℃ for tissue-specific expression analysis.
The hairy roots of S. miltiorrhiza (25℃,120rpm) were treated with 200µg/L ethephon in 21 days in liquid 6-7-V medium. The treated samples were collected at 0h,1h,2h,4h,8h,12h,24h and 72h, respectively, and the untreated S. miltiorrhiza hairy roots of under the same culture conditions were collected as controls. Each of the three biological replicates was collected and frozen in liquid nitrogen.
Cloning of SmERF1b-like gene
The sequence information of SmERF1b-like gene (MH006601.1) was obtained from our previous research[22]. The amplification primers were designed with primer6.0. The primer listed in TableS1. The cDNA of S. miltiorrhiza was used as template. A 50µL reaction system (25µL 2xM5 HiPer plus Taq HiFi PCR mix, 2µL upstream primer, 2µL downstream primer,2µL cDNA template,19µL ddH2O) was used for amplification. The reaction conditions were:95℃,5 min, 38 cycles (95℃, 30s, 55℃, 30s, 72℃,1min), 72℃,10 min. The PCR product was separated by 1% agarose gel electrophoresis, and the target gene fragment was purified and recovered by gel recovery kit (TOLOBIO). The target gene fragment was ligated into pMD19-T simple cloning vector, then transformed into DH5α. Positive clones were screened in LB solid plate (50 mg/L Amp), 37℃,12h. Then, PCR identified positive clones were sequenced.
Bioinformatics analysis of SmERF1b-like gene
The nucleotide sequence information of SmERF1b-like gene (MH006601.1) was obtained by NCBI database (http://www.ncbi.nlm.nih.gov).The amino acid sequence of SmERF1b-like gene was obtained by online program FGENESH (http://www.softberry.com/berry.phtml) and the gene structure was predicted. The characteristics of SmERF1b-like protein were analyzed by ProtParam (http://web.expasy.org/protparam/) The protein secondary structure was predicted by SOPMA (http://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). The protein tertiary structure was predicted by SWISS-MODEL (http://swissmodel.expasy.org/interactive). The signal peptide was predicted by SignalP 5.0 Serve (https://services.healthtech.dtu.dk/service.php?SignalP-5.0).
Total Dna Extraction, Total Rna Extraction And Cdna Synthesis
Total DNA extraction, total RNA extraction and cDNA synthesis
DNA was extracted from 0.1g S. miltiorrhiza sample stored at -80℃ by modified CTAB method[26]. RNA extraction kit (Tiangen, China) was used to extract complete RNA from S. miltiorrhiza samples stored at-80℃ according to the instructions. The extracted RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser kit (TaKaRa, China) according to the instructions. Total RNA was extracted and cDNA was synthesized by liquid nitrogen quick grinding of the head, stem, leaf, flower and root of S. miltiorrhiza plants stored at -80℃ and the hairy roots treated with exogenous ethephon.
Qrt-pcr Analysis
Fluorescent quantitative primers were designed according to the SmERF1b-like gene sequence, SmActin was used as an internal reference gene, Quantitative experiments were performed with a BioRad CFX Manager 3.1 real-time PCR (qRT-PCR) system according to the SYBR Premix ExTaq (TaKaRa) kit instructions, reaction conditions: 95℃,30 s;40 cycles (95℃,5 s; 60℃,30 s), The gene expression of SmERF1b-like treated with exogenous ethylene was analyzed in S. miltiorrhiza hairy roots. The primers listed in Table S.
Subcellular localization analysis of SmERF1b-like
XbaI and BamHI were selected to construct the vector using 1.2 sequencing correct bacterial solution as the template, and the primers are shown in Table S1.The target fragment containing the restriction site was amplified and ligated into the 19T vector, and the positive plasmid was obtained by sequencing screening and the PA70390-SmERF1b-like vector was constructed by double digestion using the PA70390 unloaded plasmid, and the ligation product was transformed into DH5aE.coli.The positive monoclonal antibody was selected by LB solid plate and colony PCR with Kan antibiotics (50mg/L) and the plasmid was extracted. Nicotiana benthamiana was used to inject positive Agrobacterium EHA105 containing PA70390-SmERF1b-like plasmid into tobacco leaves, which were cultured overnight in the dark at 25℃ and 12h to collect tobacco leaves to prepare protoplasts for observation by confocal laser scanning[26].
Emsa Verification
The induction and purification of MBP and SmERF1b-like protein implied as previously described[22]. The 0.4 mM IPTG was used in induced protein expression. The designed and synthesized ERF protein recognition element DNA probe (CGCCGCC) was annealed, and after cooling at room temperature, the probe and purified SmERF1b-like protein were run at room temperature for 40min using 6×loading buffer, and the colloid was placed in nucleic acid staining solution after completion and staining for 30min.After rinsing for 2–3 times, the gel was then placed under a chemiluminescence imaging system (ChemiDoc MP, Bio-Rad) for photography and preservation, and the experimental method was based on a previous report[22].
Acquisition and identification of transgenic S. miltiorrhiza hairy roots
Using the sequenced positive clone as the template, primers SmERF1b-like-Z-F/R (listed in Table S1) with the restriction site, digested and ligated with the PCAMBIA1304 vector. The overexpressed PCAMBIA1304-SmERF1b-like and antisense PCAMBIA1304-smerf1b-like vectors positive clones identified by PCR, the identify primers listed in Table S1, then the corrected clones sequenced. Then the corrected PCAMBIA1304-SmERF1b-like, PCAMBIA1304-smerf1b-like and PCAMBIA1304 plasmids were transformed into Agrobacterium rhizogenes ATCC15834 respectively. The positive clones were screened by YEB liquid plate (50mg/L Kan), 28℃,220rpm cultivated, then PCR identified. The identified primer listed in Table S1. The positive clones were used for downstream experiments. PCAMBIA1304, PCAMBIA1304-SmERF1b-like and PCAMBIA1304-smerf1b-like plasmid positive bacteria were used to induce S. miltiorrhiza hairy roots. The detailed steps referred to the previous described[22]. DNA extracted according to the instructions of the Plant Genomic DNA Extraction Kit (Tiangen, China) and identified using HptII, rolb, rolc and transgene identification primers, and the primer sequences are shown in Table S.
The S. miltiorrhiza hairy roots were collected and dried at 45℃ for 3 days, ground, weighed and stored in 2mL EP tube.0.04g dry powder was added to 8mL of 70% methanol, at room temperature for 8 h. Then 8000rpm, centrifugation 10minutes.Tanshinone compounds were detected by HPLC as previous described after[26].
Data analysis
Statistical analyses of HPLC data and qRT-PCR data were performed using one-way analysis of variance in SPSS (version 13.0) and P < 0.05 was considered statistically significant. All images were generated using EXCEL (version 2010) and TBtools software.