Functional validation of enzymes in Salvinorin A biosynthesis - LCMS data.

Expression of genes in N. benthamianaRecombinant constructs of pTRBO and pEAQ vectors containing either full-length or truncated coding sequences of genes (AtHMGR, TccytGGPPS, SspKPS, SspKLS, SdANS, SdHDAS, SdDAS, SdCMT) from Arabidopsis thaliana, Taxus canadensis, Salvia splendens, and Salvia divinorum were introduced into Agrobacterium tumefaciens strain GV3101 (Weidi Biotechnology, Shanghai) and then infiltrated into N. benthamiana. Positive colonies of transformed Agrobacterium tumefaciens were identified by PCR and subsequently cultured in 10 ml LB medium with rifampicin, gentamycin, and kanamycin for 2 days at 200 rpm and 28℃. After centrifugation at 4000 rpm for 15 minutes, the cells were resuspended in 1× MMA (10 mM MgCl2, 10 mM MES, 150 mM acetosyringone) and adjusted to an OD600 of 0.2, then kept in the dark for at least 1 hour. Agrobacterium strains containing pTRBO or pEAQ plasmids were mixed for co-infiltration into leaves of N. benthamiana. The Agrobacterium suspensions were then infiltrated into the leaves of 4-6-week-old healthy N. benthamiana plants. For the SdCMT products, SAM (S-adenosyl methionine) was infiltrated into the plants on day six. On day seven, the infiltrated leaves were collected, ground with a pestle and mortar, and extracted with ethyl acetate overnight at 28℃ while shaking at 220 rpm. The samples were centrifuged, and the supernatant was transferred to a new glass tube, dried using a Rotary Evaporator, and resuspended in 1 mL methanol, with around 100 µL aliquoted for LC-MS analysis.LC-MS analyses of N. benthamiana extracts.The ethyl acetate extracts of N. benthamiana were concentrated using a rotary evaporator, then dissolved in methanol for LC-MS analysis (SCIEX Zeno TOF 7600, SCIEX) on a Kinetex 2.6 µm C18 100 Å column (100 x 2.1 mm). The aqueous phase (Phase A) consisted of 2 mM ammonium formate with 0.01% formic acid, while the organic phase (Phase B) was acetonitrile. The gradient conditions were as follows: increase Phase B from 0% to 10% over 0 to 0.5 minutes, from 10% to 40% from 0.5 to 1 minute, then ramp up to 99% from 1 to 8 minutes, holding at 99% for an additional 2 minutes.