Bl21 codon plus

To examine the enzymatic function of NaTPS25 in vitro, a truncated version of NaTPS25_AZ (missing the first 13 codons) was cloned from cDNA of Arizona genotype plants using the primer pair TPS25f/r (Table S2). The resulting PCR fragment was cloned into the pET200/D-TOPO vector (Invitrogen, Carlsbad, CA, USA) and the Escherichia coli strain BL21 Codon Plus (Invitrogen) was used for heterologous expression. The protein expression and bacterial extract preparation were done as described in a previous study (Zhou et al., 2017) . Enzyme assays were performed in a Teflon ® -sealed, screw-capped 1 ml GC glass vial containing 50 µl of the bacterial extract and 50 µl assay buffer with 10 µM geranyl diphosphate, 10 mM MgCl2, 0.2 mM Na2WO4 and 0.1 mM NaF. A solid phase micro-extraction (SPME) fiber coated with a 100 µm-thick layer of polydimethylsiloxane (SUPELCO, Belafonte, PA, USA) was placed into the headspace of the vial where it was incubated for 60 min at 30°C, before it was inserted into the injector of the gas chromatographer to analyze the absorbed reaction products. GC-MS analysis was conducted using an Agilent 6890 Series gas chromatographer coupled to an Agilent 5973 quadrupole mass selective detector. The GC was operated with a DB-5MS column (Agilent, Santa Clara, USA, 30 m x 0.25 mm x 0.25 µm).