Plasmid miniprep kit

DNA restriction enzymes, DNA Ligation high, Taq DNA polymerase, DNA marker, and protein marker were purchased from the Beyotime Institiute of Biotechnology (Shanghai, China). The Gel Extraction kit and Plasmid Miniprep kit were purchased from TIANGEN biotech (Beijing) Co., Ltd., (Beijing, China). Ni Sepharose™ 6 Fast Flow was purchased from GE Healthcare (Fairfield, MA, USA).
Ethyl acetate, n-butanol, and methanol were bought from Macklin (AR, Shanghai Macklin Biochemical Co. Ltd., Shanghai, China). NaCl, HCl, NaOH (AR, China National Pharmaceutical Group Corporation, Beijing, China), and distilled water were used. Chromatographic-grade methanol from Fisher (HPLC, Fisher Scientific, Waltham, MA, USA) was used for high performance liquid chromatography (HPLC) analysis. Paeoniflorin standards were purchased from YuanYe company (Shanghai, China).

Polymerase chain reaction (PCR) was performed to amplify a 651bp sequence surrounding rs13005431 from genotyped genomic DNA with the TT genotype of rs13005431. Primers were designed using Primer-BLAST (www.ncbi.nlm.nih.gov/tools/primer-blast/) and sequences for restriction sites NheI and XhoI were introduced (Supplementary Table 2). Both gel purified PCR product and pGL3-promoter reporter vector (Promega, Madison, WI, USA) were digested by NheI and XhoI restriction enzymes (Takara Bio Inc., Kusatsu, Japan) at 37°C for 4 hours and then ligated by T4 DNA ligase (TaKara Bio Inc.) at 16°C for 8 hours. After transforming into competent E. coli DH5α cells and extraction by plasmid miniprep kit (TianGen, Beijing, China), the recombinant plasmid vector was verified by direct sequencing and named PGL3-rs13005431T-promoter (Supplementary Figure 1). A site-directed mutagenesis strategy was applied to acquire another recombinant plasmid vector of PGL3-rs13005431C-promoter (see Supplementary Table 2 for primer sequences) which was confirmed by DNA sequencing.

Plasmids constructed in this study harbouring an ITS1 gene fragment were extracted from Escherichia coli hosts using a plasmid miniprep kit (Tiangen, China). After extraction, plasmid DNA was sequenced and aligned to the NICB database, resulting in a 100% fungal lineage match (GenBank MN044804.1). Plasmid DNA concentration was measured with a Nanodrop 2000 spectrophotometer (Thermo, USA). The plasmid copy number was 8.50 × 10⁷ copies∙μl⁻¹ and standard curves spanned a range from 4.25 × 10² to 4.25 × 10⁶ copies∙μl⁻¹. Standard reactions were performed for all samples in triplicate using an ABI 7500 sequence detection system (Applied Biosystems, Canada) using the SYBR green quantitative PCR (qPCR) method. Each qPCR mixture (25 μl) comprised 12.5 μl of Maxima SYBR green/Rox qPCR master mix (Fermentas, Lithuania), 1 μl of each primer (10 μM), 5 μl of template DNA, and ddH₂O (5.5 μl). All reactions were performed in 8-strip thin-well PCR tubes equipped with ultraclean cap strips (ABgene, UK). The specificity of qPCR amplification was determined by melting curve and gel electrophoresis analyses. In all experiments, the same procedure was applied to negative controls without template DNA to eliminate contamination. The abundance of each gene was calculated based on the resulting standard curves and converted to copies per gram of soil, assuming a DNA extraction efficiency of 100%.