Wizard miniprep kit

The genomic DNA was isolated from electroporated cells using a DNeasy Blood & Tissue Kit (Qiagen). The junction of the target gene and the reporter gene was amplified by PCR and amplicons were subcloned into TOPO cloning vector using the Zero Blunt TOPO Kit (Invitrogen). Clones of amplicons were isolated from each Escherichia coli colony using a Wizard Miniprep Kit (Promega) and sequenced using M13F and M13R primers. 

Plasmids, shown in Supplementary Table S2, were constructed by Gibson Assembly. Gibson assembly primers were designed using NEBuilder version 2.5.2 (https://nebuilder.neb.com/#!/ accessed on 14 March 2021). DNA was amplified from wild-type genomic DNA by PCR using Advantage 2 Polymerase (Cat#639201; Takara Bio USA, Mountain View, CA, USA). Plasmid backbones were double-digested with the necessary restriction enzymes and purified using the Qiagen DNA purification kit (QIAGEN GmbH, Hilden, Germany). DNA inserts were cloned into the digested vectors using the NEBuilder Hifi DNA Assembly Mastermix (E2621S; New England Biolabs, Ipswich, MA, USA) and incubated for 1 h at 50 °C. Newly-assembled plasmids were transformed into GC10 E. coli cells made chemically-competent. Then, 2 μL of the Gibson Assembly mixture was added to the 50 μL GC10 competent cells, placed on ice for 30 min, heat shocked for 30 s at 42 °C, placed on ice for 2 min, and then 950 μL of S.O.C. medium was added. The mixture was incubated at 37 °C for 1 h shaking at 250 rpm and then plated on LB plates with the appropriate antibiotic. Colonies were screened by colony PCR, and positive colonies were cultured in 5 mL of LB + antibiotic overnight. Plasmids were purified using the Wizard Miniprep Kit (Promega Corporation, Madison, WI, USA) and sequenced by Eton Bioscience.

Genomic DNA was isolated from closed buds according to Aldrich and Cullis (1993 (link)). The 5′-flanking regions were isolated using the GenomeWalker™ Universal Kit (Clontech, Saint-Germain-en-Laye, France) according to the manufacturer’s instructions. The first primer pair was designed based on the F3′H sequence (GenBank: FJ216426) (Schlangen et al. 2010 (link)). DNA-fragments were isolated and ligated into the vector pCR^®2.1-TOPO (Invitrogen, Paisley, UK) and transformed in E. coli TOP10 (Invitrogen, Paisley, UK). Plasmids were isolated using the Wizard Miniprep Kit (Promega, Mannheim, Germany) and sequenced by StarSEQ (Mainz, Germany). Further primers for genome walking were designed from the sequences obtained (Suppl. Table S1). Finally, a putative F3′H promoter sequence containing 1712 bp of the 5′-flanking region (GenBank: KU508433) was obtained with the primer pair Pro.F3′H. A putative chalcone 3-hydroxylase (CH3H) promoter sequence was obtained in a similar way (GenBank: KU508432) (Supplemental material).
The transcription start site (TSS) in the 5′-flanking region of the F3′H was predicted by using the TSSP software (Softberry, http://linux1.softberry.com), and the cis-acting regulatory elements were analysed by using the PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Chemically competent E. coli cells (100 μl) were thawed on ice and transferred into a chilled 14 mL polypropylene round-bottom tube (BD Biosciences). Then 50ng of the desired plasmid DNA was added to the E. coli and the E. coli/DNA mixture was incubated for 20–30 min on ice. Subsequently, this E. coli/DNA mix was heat-shocked for 40 seconds in a 42°C water bath and cooled on ice for 2 min. S.O.C medium (250 μl, Life Technologies) was added and tubes shaken at 200 rpm for 1 h at 37°C. The transformed E. coli were plated onto Lysogeny Broth (LB) agar plates with the appropriate antibiotics and incubated overnight at 37°C. For subsequent analysis of clones obtained, single colonies were picked and inoculated in LB medium containing ampicyline, incubated overnight at 37°C and plasmid DNAs were purified using the Promega Wizard mini-prep kit.

A P. pastoris codon optimised nucleotide sequence (https://eu.idtdna.com/CodonOpt) encoding PA1 without the signal peptide (NCBI Accession P62930 residues 33–103), referred to as pea albumin full (PAF), and cloning primers were purchased from Integrated DNA Technologies (IDT). Restriction endonucleases were supplied by Thermo scientific or New England BioLabs. Electrophoresed DNA fragments were purified from excised gel slices using a Qiagen gel extraction kit. Plasmid DNA was prepared using Promega Wizard miniprep kits. T4 ligase kit was supplied by Promega. Phusion polymerase was from New England Biolabs. Pichia pastoris (SMD1168H strain), the expression vector pGAPZαB, and Easy comp Pichia transformation kit were from Invitrogen.
Anti-GNA antibodies were prepared by Genosys Biotechnologies, Cambridge, UK. Monoclonal 6x-His Tag Antibodies were from Fisher Scientific, UK. Secondary IgG horseradish peroxidase antibodies were from Biorad. Chemicals for chemiluminescence and buffer salts were supplied by Sigma.