Cloning and Characterizing JcTAW1 Gene

JcTAW1 gene sequence was retrieved with the query of rice TAW1 protein from the whole-genome shotgun contigs (WGS) of Jatropha via the tblastn tool of NCBI. Similar to the rice TAW1 gene, JcTAW1 has no introns, so it can be amplified directly from Jatropha genomic DNA extracted as previously described [26 (link)]. JcTAW1 gene was amplified by two rounds of nested PCR using Phusion high-fidelity DNA polymerase (NEB). The first round of PCR used the flanking gene primers JcTAW-Fw, JcTAW-Rv and Jatropha genomic DNA as the template. The crude product was 100-fold diluted as a template for the second round of PCR with the gene-specific primer pair JcTAW1-5Kn/JcTAW1-3Sc. Then the purified amplicon fragment was cloned into the plant binary vector pBI121 (Clontech) by KpnI and SacI (NEB) digestions to generate plant expression vector pBI121 (JcTWA1). All primers used for vector construction are listed in Table S1.