| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The final PCR product containing partial IRES sequence and 3xFLAG was digested by KpnI and BamHI and ligated to corresponding restriction sites in pT7CFE1-NHa. Synthetic PaCPT1 and PaCPT2 (GenScript, NJ, USA) were ligated to BamHI and XhoI sites in pT7CFE1-3xFLAG. PaCPT3 was amplified from P. argentatum leaf cDNA using a primer set, 27/28. The PaCPT3 PCR product was cloned into pT7CFE1-3xFLAG via BamHI and XhoI restriction sites. Synthetic PaCBP (GenScript, NJ, USA) was amplified using a primer set 29/30 and digested with BamHI and XhoI, followed by ligation to corresponding sites in pT7CFE1-NHa. A control construct, pT7CFE1-NHa-GFP for co-immunoprecipitation described below, was generated by cloning GFP sequence into pT7CFE1-NHa. GFP was amplified from pCFE-GFP (ThermoFisher Scientific) using a primer set 31/32, containing BamHI and XhoI, respectively, followed by ligation to corresponding sites in pT7CFE1-NHa. The purified plasmid preparations of all IVT constructs were cleaned up further by ethanol precipitation, followed by resuspension in nuclease-free water to a concentration of 500 ng μL-1 prior to IVT. IVT was carried out using 1-Step Human Coupled IVT Kit (ThermoFisher Scientific), following the manufacturer’s procedure except for reducing the reaction to half of the recommended amounts. | Get A Quote |
Guayule (Parthenium argentatum) is a perennial shrub in the Asteraceae family and synthesizes a high quality, hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR). Despite its potential to be an alternative NR supplier, the enzymes for cis-polyisoprene biosynthesis have not been comprehensively studied in guayule. Recently, implications of the protein complex involving cis-prenyltransferases (CPTs) and CPT-binding proteins (CBPs) in NR biosynthesis were shown in lettuce and dandelion, but such protein complexes have yet to be examined in guayule. Here we identified four guayule genes – three PaCPTs (PaCPT1-3) and one PaCBP, whose protein products form PaCPT/PaCBP complexes. Co-expression of both... More
