We had previously amplified the AaHMGR (AF142473), AaFPS (AF112881), and AaDBR2 (PWA95605.1) Open Reading Frames (Wang et al., 2011 ; Shen et al., 2018 (link)). PCR products were separated on 1% Agarose gel electrophoresis and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). Purified PCR products were then cloned into the pJET2.1 vector (Promega), transformed into DH5α high-efficiency competent cells, and grown on LB medium supplemented with Carbenicillin (Cb). Three positive colonies were subjected to colony PCR and submitted for sequence analysis (Sangon sequencing company, Shanghai). For sequence alignment, the DNAMAN software (version 5.6) was utilized. EPSPS was substituted for the hygromycin gene in the XhoI restriction site of pCAMBIA1305. The vector pCAMBIA1305.1-EPSPS-HMGR was created by inserting 35S-HMGR-nos into the HindIII and EcoRI restriction sites of pCAMBIA1305-EPSPS using 5× In-fusion HD Enzyme Primix (Clontech, Dalian, China). The FPS-nos-35S-DBR2 fragment was then inserted into the NcoI and BstEII restriction sites of pCAMBIA1305.1-EPSPS-HMGR (Figure 2A).
The full-length CDS for ORA (JQ797708) was amplified and digested with BamHI and SacI. The full-length ORF was then cloned into the BamHI and SacI sites of the pCAMBIA2300+ vector under the CYP71AV1 promoter to create pCAMBIA2300-proCYP71AV1::AaORA::NOS (Figure 2B).
The ORF of AaHD1 (KU744599), was amplified and cloned into the PHB vector (Figure 2C). After validating the transformation, the positive colonies were transferred into Agrobacterium tumefaciens strain EHA105 and inoculated into A. annua.
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