Escherichia coli

The PAL open reading frame was incorporated into the pET-32a vector (TaKaRa, Beijing, China) and transferred into Escherichia coli (TaKaRa, Beijing, China) to express the recombinant protein (PAL-B, PAL-H, Table S1). Protein expression was triggered by the addition of isopropyl-β-D-thiogalactoside (IPTG, TIANGEN, Beijing, China) to an ultimate 1 mmol/L concentration and keeping the temperature at 18 °C for 10 h. The Capturem His-Tagged Purification Miniprep Kit (TaKaRa, Beijing, China)was used to purify the PAL protein. The results were observed by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The reaction conditions for verifying the catalytic function of PAL were as follows: 2 mL 0.1 mol/L borate buffer (pH 8.8); 1 mL 0.02 mol/L L-phenylalanine (pre-dissolved in borate buffer); 100 μL purified protein (100 μL borate buffer was used as a blank control); water bath at 30 °C for 30 min. The reaction liquid was analyzed for the formation of trans-cinnamic acid using liquid chromatography (Waters 1525) with a C18 column (150 × 4.6 mm, 5 μm).

DNA was extracted from the leaf tissue (two- to three-leaf stage) of untreated S plants and R surviving 15.8 g mesosulfuron-methyl ha⁻¹ (10 plants per population) using the CTAB method [34 (link)]. The open reading frame (ORF) regions of the ALS gene from the S and R plants were cloned using the primer pair (F: 5′-GGTGCATCAATGGAGATTCA-3′, R: 5′-TCAGTATTTAGTCCGGCCATC-3′) designed based on the sequence of the shepherd’s-purse ALS gene (NCBI ID: HQ880660.1). Polymerase chain reaction (PCR) was conducted in a 25 μL volume, consisting of 2 μL DNA, 0.5 mmol L⁻¹ of each primer and 12.5 μL of EmeraldAmp PCR Master Mix (Takara Bio Inc., Shiga, Japan), running with the following steps: 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s, followed by a final extension of 7 min at 72 °C. The PCR product was purified from agarose gel (1%) using a FastPure Gel DNA Extraction Mini Kit (Vazyme Co., Ltd., Nanjing, Jiangsu, China). The purified fragment was then cloned into the pMD19-T vector (Takara) and transformed to Escherichia coli (Takara). Five white colonies from each sample were selected for sequencing. Sequencing results were analyzed by DNAMAN 8.0 (Lynnon Corp., Quebec, QC, Canada).