Vector Construction and Transformation of Artemisia annua

For AaEIN3-overexpression vector construction, the coding region of AaEIN3 with a BamHI and a PstI restriction site on either end, respectively, was ligated into the pHB+ vector under the control of the CaMV35S promoter to generate pHB-35S:sGFP-AaEIN3:Noster construct, with the sGFP fused to the N-terminal of AaEIN3. For RNA interference (RNAi) vector construction, the primers AaEIN3-RiF (5′-CACCTGAATCGTGGCGGAACGCTAAA-3′) and AaEIN3-RiR (5′-ACTGAAACCCTGCTGGCATAAA-3′) were designed to amplify a 350 bp-long RNAi fragment with cDNA of AaEIN3 as the template. By using the gateway cloning system, the RNAi fragment was first ligated into TOPO vector and then cloned into the pHellsGate12 vector via LR reaction (Invitrogen, United States) to generate the final pHellsGate-RNAi construct. The resulting AaEIN3-overexpression and RNAi constructs were transduced into Agrobacterium tumefaciens strain EHA105, respectively, and then introduced into Artemisia annua to generate transgenic Artemisia annua plants as previously described (Shen et al., 2012). Independent transgenic lines were grown and selected in hygromycin-containing MS medium for pHB-AaEIN3 overexpression transformants, and in kanamycin-containing MS medium for pHellsGate-RNAi transformants. AaEIN3-overexpression plant lines were confirmed by PCR detection with primers AaEIN3-ORF1 (5′-GGATCCATGGGGATGGGGATCTTTGAAG-3′) and rbc48-A (5′-GCATTGAACTTGACGAACGTTGTCGA-3′). And AaEIN3-RNAi lines were confirmed by PCR detection with primers p35S-FP (5′-TTCGTCAACATGGTGGAGCA-3′) and AaEIN3-RiR (5′-ACTGAAACCCTGCTGGCATAAA-3′). These transgenic lines were transferred to soil and kept for further analyses.