Platinum superfi 2 polymerase

For gene cloning, full-length GmC4Hs were amplified from soybean cDNA using gene-specific primers (S1 Table) and Platinum SuperFi II Polymerase (Invitrogen, USA). The PCR products were cloned into the gateway entry vector pDONR-Zeo (Invitrogen, USA) using the BP clonase reaction mix (Invitrogen, USA), transformed into E. coli DH5α, and grown on LB media supplemented with zeocin (50 μg/mL). The E. coli colonies containing recombinant plasmids were screened by colony PCR using gene-specific primers (S1 Table) to get pDONRZ-GmC4H. The entry clones were sequenced for confirmation followed by recombination into pESC-Leu2d-LjCPR1-GW using LR clonase reaction mix (Invitrogen, USA) for enzyme assay or pEarleyGate101 for subcellular localization. The recombinant plasmids were transformed into E. coli DH5α, screened by colony PCR using gene-specific primers. Positive pESC-Leu2d-LjCPR1-GW-GmC4H plasmid DNA was transformed into Saccharomyces cerevisiae strain BY4742 using Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA). Positive pEarleyGate101-GmC4H plasmid DNA was transformed into Agrobacterium tumefaciens GV3101 for subcellular localization.

A list of constructs used in this study is provided in Supplementary Material. For cloning, PCR fragments were amplified using Platinum SuperFi II Polymerase (Invitrogen). The fragments were assembled by either NEBuilder HiFi Assembly (New England Biolabs, NEB) or TOPO directional cloning (Invitrogen). Assembled products were heat-shock transformed to 5-alpha (NEB), TOP10 (Invitrogen), or BL21 Star™ (DE3) (Invitrogen) bacteria.