Gateway vector pcr8 gw topo

AlySEP3 with upstream region was amplified using primers Fw 5′-CTTGACTAGCCCACAACACTTC-3′ and R 5′-AATAGAGTTGGTGTCATAAGGTAACC-3′. The polymerase chain reaction fragments were cloned into the GATEWAY vector pCR8/GW/TOPO from Invitrogen and transferred through LR reaction into the destination vector AM884381 (pGREEN-GW-eGFP; Zhong et al. 2008 (link)). Expression vector was introduced into A. thaliana ecotype Col-0 by floral dip transformation. Transformant plants were selected on MS medium with BASTA. For comparison, we used previously generated pSEP3::SEP3-GFP plants (4.1 kb promoter; Smaczniak et al. 2012 (link)).

The full lengths of PgDDS (GenBank accession number AB122080), CYP716A47 (GenBank accession number JN604536), and CYP716A53v2 (GenBank accession number JX036031) used in this study were isolated by PCR. The primers used for isolation of the open reading frame sequences of PgDDS, CYP716A47, and CYP716A53v2 are follows. The forward primer to amplify the PgDDS gene was 5′- ATG TGG AAG CTG AAG GTT GCT CAA GGA-3′, and the reverse primer used was 5′- TTA AAT TTT GAG CTG CTG GTG CTT AGG C-3′. The forward primer used for the CYP716A47 gene was 5′- ATG GTG TTG TTT TTC TCC CTA TCT-3′, and the reverse primer was 5′- TTA ATT GTG GGG ATG TAG ATG AAT-3'. The forward primer used for CYP716A53v2 was 5′- ATG GAT CTC TTT ATC TCA TCT CAA-3′, and the reverse primer was 5′- TTA AAG CGT ACA AGG TGA TAG ACG-3′. To generate overexpression, the open reading frame sequences of these genes were cloned into GATEWAY vector pCR8/GW/TOPO (Invitrogen, Carlsbad, CA) and then transferred to the pZIP-Bar destination vector, in which PgDDS, CYP716A47, and CYP716A53v2 were driven by the 35S Cauliflower mosaic virus (CaMV) promoter. The vector was inserted into competent cells of Agrobacterium tumefaciens GV3101 and transformed into tobacco plants by using the same methods as in the study by Han et al [33] (link).