Genetic Engineering of Poplar Auxin Pathway

The coding sequences of PtrFBL1, 2, 3, 4, 5, 6, 7, and 8, without stop codons, were amplified from the cDNA of hybrid poplar 84K (Populus alba × Populus glandulosa) and inserted into pEarleyGate101 (ABRC stock DB3-683) to produce the 35S::PtrFBL1-YFP, 35S::PtrFBL2-YFP, 35S::PtrFBL3-YFP, 35S::PtrFBL4-YFP, 35S::PtrFBL5-YFP, 35S::PtrFBL6-YFP, 35S::PtrFBL7-YFP, and 35S::PtrFBL8-YFP constructs, respectively, using the Gateway cloning system (Invitrogen). The approximately 2.5 kb 5′-UTR fragments of PtrFBL1, PtrFBL4, PtrFBL5, and PtrFBL7 were amplified from the genomic DNA of P. trichocarpa Torr. The primer sequences for the promoters are listed in Table S1. The promoter fragments were then inserted into pDNOR222.1 and subcloned into pMDC164 to produce P_PtrFBL1::GUS, P_PtrFBL4::GUS, P_PtrFBL5::GUS, and P_PtrFBL7::GUS constructs using the Gateway cloning system (Invitrogen). The coding sequence of PtrFBL1 was amplified from the cDNA of 84K, cloned into pDNOR222.1 and sequenced. PtrFBL1 cDNA was further cloned into pMDC32 to produce 35S::PtrFBL1 constructs for transformation into poplar 84K. The transgenic poplar plants containing the auxin responsive promoter DR5 were obtained previously (Liu et al., 2014a (link)). At least five independent transgenic lines were used for further analyses.