Salvia miltiorrhiza Bunge

5.1. Identification and phylogenetic analysis of the NAC family genes in S. miltiorrhiza The assembly and annotation data of Salvia miltiorrhiza Bunge (Lamiaceae) in the Genome Warehouse in BIG Data Center under Project numbers PRJCA003150, which are accessible at https://bigd.big.ac.cn/ gwh (Song et al., 2020). The Arabidopsis NAC amino acid sequences were obtained from TAIR (http://www.arabidopsis.org) and were used as query in searches against the S. miltiorrhiza genome database using the BLASTP program to obtain homologous sequences (Jin et al., 2020). The Hidden Markov Model (HMM) corresponding to the NAC domain (PF02365) was downloaded from the pfam protein database, and HMMER 3.2 was used to examine the NAC genes from the BLASTP aligned sequences. Default parameters were employed, and the cutoff value was set to 0.01 (Letunic and Bork 2018). Genes encoding proteins containing NAC domains were identified as NAC genes. All the Sm -NACs were mapped to the eight chromosomes and one scaffold of S. miltiorrhiza using the TBtools program and the physical locational information from the S. miltiorrhiza genome (Li et al., 2020a). A multi-sequence alignment of NAC proteins from Arabidopsis and S. miltiorrhiza was performed using ClustalW in MEGA7.0 with default parameters (https://www.megasoftware.net/). Because the Sm-NAC family sequence lengths varied greatly, the alignment results were used to construct a phylogenetic tree using the Maximum Likelihood method with 1000 bootstrap replicates (Felsenstein 1985; Jones et al., 1992). Additionally, Evolview (http://www.evolgenius.info/) was used to beautify the evolutionary tree (Subramanian et al., 2019). 5.2. Gene structure and protein motif analyses of Sm-NAC genes An online program of the gene structure display server (GSDS2.0) (http://gsds.cbi.pku.edu.cn/index.php) was used to draw the exonintron distribution of each Sm - NAC gene by comparing predicted coding sequences (Hu et al., 2015). Conserved motifs of Sm-NAC protein sequences were investigated using the online software MEME5.0.4 (htt p://meme-suite.org/tools/meme) with default values for the motif parameters and the number of motifs searched set as 20 (Bailey et al., 2009; Munir et al., 2020). TBtools was used to visualize the results (Chen et al., 2020). 5.3. Expression profile analysis using transcriptome data To understand NAC gene expression changes after MeJA exposure in S. miltiorrhiza and the expression levels in different tissue, the transcriptome data was retrieved from the NCBI Sequence Read Archive. For the different tissues, we selected three tissues in the same batch of Sequence Read Archive (SRA) data: flower, leaf, and root (accession numbers: SRR1020591, SRR1043998, and SRR1045051) (Chen et al., 2014). The transcriptome data after the MeJA treatment was selected from two-month-old sterile seedlings grown on 0.5 × MS medium containing 100 μM MeJA or the simulated solution (ethanol). The roots of the treated seedlings were collected from three biological replicates (accession numbers: SRR11484256-SRR11484259, SRR11484266, and SRR11484271) (Zhou et al., 2020). In accordance with the TopHat BAM files and the reference GTF file, cuffdiff was used to calculate fragments per kb per million reads values (FPKM) of different tissue samples and MeJA-treated samples, and the expression differences between different samples were determined at the same time (Sulayman et al., 2019). 5.4. RNA isolation and qRT-PCR Total RNA was extracted from the rhizome, leaves, and transgenic roots of S. miltiorrhiza in accordance with the instructions of the polysaccharide and polyphenol plant RNAprep Pure Plant Kit (TIANGEN, China). The RNA from the rhizome, leaves, and transgenic roots of S. miltiorrhiza were mixed and reverse transcribed into cDNA using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The cDNA was used as the template to amplify the NAC 2 gene for cloning. The cDNA for qRT-PCR was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Tokyo, Japan) with oligo dT. The qRT-PCR was performed in accordance with the instructions of the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa on a QuantStudio™ 6 Flex (Life Technologies, Carlsbad, CA, USA)). The procedure was as follows: 95 ◦ C for 30 s, then 40 cycles of 95 ◦ C for 5 s and 59 ◦ C for 30 s. Each reaction was repeated three times. The reference gene was the actin (Yang et al., 2010). The primers used are list in Supplementary Table S2. The 2 ΔΔ Ct method was used to analyze the qRT-PCR data and calculate the relative expression levels (Livak and Schmittgen, 2001). 5.5. Plant expression vector construction To construct the Sm - NAC 2 -overexpression vector, the gene-specific primers Sm-NAC2-OE-F and Sm-NAC2-OE-R were used to amplify the complete ORF of Sm - NAC 2. Gateway technology was used to construct the expression vector. First, the ORF of Sm - NAC 2 was cloned into the pDONR207 entry vector using the BP Clonase Enzyme Kit, and then, it was cloned into the pK7WG2R destination vector using an LR Clonase Enzyme Kit (Invitrogen, MA, USA) (Ding et al., 2017). A 116-bp sequence was amplified using the primers Sm-NAC2-RNAi- F and Sm-NAC2-RNAi-R to construct the plant RNAi vector. The amplified fragment was cloned into the pDONR207 entry vector, and then cloned into the pK7GWIWG2R binary vector, as described by (Ding et al., 2017). The recombinant vector was confirmed by sequencing. The primers used in this experiment are provided in Supplementary Table S2. 5.6. Acquisition of S. miltiorrhiza transgenic transgenic roots The leaves of the sterile seedlings of S. miltiorrhiza were cut into small pieces of 1 × 1 cm and placed them on a 1/2MS solid medium for cultivating in the dark for 2–3 days at 25 ◦ C. Single colonies of the A. rhizogenes cells harboring the recombinant plasmid were inoculated into 50 ml of liquid YEB medium with 50 mg l 1 of spectinomycin, and grown on a shaker at 28 ◦ C for 16–18 h until the OD 600 nm reached 0.6. Cells were collected by centrifugation, and re-suspended in 50 ml of liquid 1/2MS medium. Next, the leaf discs were submerged and shaken in the suspension for 30 min with 100 rpm at 25 ◦ C. Then, the leaf discs were taken out and cultured on 1/2 MS solid medium for 3 d in the dark. The leaf discs were moved onto 1/2 MS selection solid medium with 50 mg l 1 kanamycin and reduced cefotaxime. The sterilization medium was changed once in 10–15 d, and the concentration of cefotaxime in the medium was gradually reduced: from 500 mg l 1 to zero. When the transgenic roots grew to 4–5 cm, and a single root was cut from the leaf and placed on a sterile medium for individual culture. The rapidly growing kanamycin-resistant and agrobacterium-free transgenic roots were transferred to 50 ml of liquid 1/2 MS medium and maintained by transferring 0.3 g of root material into fresh 1/2 MS medium every 30 d (Ru et al., 2016). The WT control was transgenic roots developed using A. rhizogenes ATCC15834 not harboring the plasmid. The genomic DNA from fresh transgenic roots was isolated use the cetyltrimethylammonium bromide (CTAB) method (Sambrock and Russel 2001). Four pairs of specific primers were used to identify positive transgenic strains (Supplementary Table S2). The identified transgenic transgenic roots were cultured as described previously to further study Sm -NAC2 functions (Zhang et al., 2020). 5.7. Extraction and determination of tanshinones The sampled transgenic roots were placed in an oven at 45 ◦ C until they were completely dehydrated, and then, the dried S. miltiorrhiza samples were crushed into a powder using a grinder. In total, 0.02 g of sample powder was placed into 2 mL of 70% methanol. After soaking overnight in the dark, the sample was subjected to ultrasound for 45 min and then centrifuged at 8000 g for 10 min. The supernatant was removed and filtered through a 0.45 μm membrane. Afterward, 10 μL of the sample was used for HPLC detection on a Waters HPLC e2695system (Waters, Milford, MA, USA). The HPLC conditions were those established previously in our laboratory (Zhang et al., 2020). 5.8. Data statistics and analysis All the experiments were performed three times, and summary statistics are presented as means ± standard deviations (SDs). One-way ANOVAs (followed by a Tukey’ s comparisons) were used to test for significant differences among the means (indicated by different letters at P