Universal genome walker kit

The ZCT1 promoter sequence was obtained with the Universal Genome Walker Kit (Clontech). Approximately 800 bp of the ZCT1 promoter was amplified from C. roseus (Little Bright Eye, NEseeds) genomic DNA using the GSP1 and GSP2 gene‐specific primers (Table S1) matching the ZCT1 coding sequence (GenBank accession AJ632082). This sequence matches the sequence 5′ of the ZCT1 coding region of the published genomes (Franke et al., 2019; Kellner et al., 2015). The promoters of C. roseus ZCT2 and ZCT3 were obtained from the published genomes (Figure S1 and Supplemental Materials).
To identify the transcriptional start site, the SMARTer RACE cDNA Amplification Kit (Clontech) was used to amplify the 5′ cDNA ends using the same GSP1 and GSP2 gene‐specific primers. Promoter sequences were analyzed with the Plant Cis‐Acting Regulatory Elements (PlantCARE; Lescot et al., 2002) and PlantPAN 3.0 databases (Chow et al., 2019). Using the A. thaliana PlantPAN 3.0 database, only exactly matching motifs (similar score of 1) were further considered. Motifs with low information content (e.g., GAT, TF_motif_seq_0237) and frequent occurrences in all tested promoters were excluded. The pyrimidine box matches sequences from the literature (Skriver, Olsen, Rogers, & Mundy, 1991; Rogers, Lanahan, & Rogers, 1994; Gubler et al., 1999).

Upstream regulatory fragments of three monoterpene biosynthesis genes, including PbGDPS, PbTPS5, and PbTPS10, were isolated from P. bellina genomic DNA by use of the Universal GenomeWalker Kit (Clontech, USA) as previously described (Hsu et al. 2014 (link)). In addition, we also isolated 1.5-kb upstream regulatory fragments of the identified six TFs involved in the regulation of floral monoterpene biosynthesis in P. bellina (Chuang et al. unpublished) from P. equestris genomic sequences (Cai et al. 2015 (link)). The cis-elements related to light signaling and circadian clock on the upstream regulatory fragments of both monoterpene biosynthesis genes and TFs were predicted by using PlantPAN (Chow et al. 2015 (link)). The predicted results with 100% similar score were accepted.

The 5’ and 3’ RACE were carried out using the 5’/3’ RACE Kit, 2nd Generation (Roche Applied Science, Amadora, Portugal) according to the manufacturer’s instructions. Conditions for PCR were as follows: 94 °C for 2 min, 94 °C for 15 s, 59 °C for 30 s, 72 °C for 40 s, for 10 cycles; 94 °C for 15 s, 59 °C for 30 s, 72 °C for 40 s (plus 20 s/cycle), for 25 cycles, with a final elongation at 72 °C for 7 min. When necessary, a second PCR amplification was performed using the same conditions for an additional 30 cycles. To increase sequence coverage and obtain possible promoter regions, amplifications of genomic DNA were performed using the Universal Genome Walker Kit (Clontech, MountainView, CA, USA), according to the manufacturer’s instructions. Amplification products were run on agarose gels, relevant fragments purified, cloned, and sequenced as previously described.