Transcriptional Analysis of Artemisinin Pathway Genes

The RNA samples were isolated by RNA Extraction Kit (Tiangen) and the first-strand cDNA was synthesized from 2 mg of RNA using PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRA, Kusatsu, Japan). The selected pathway genes were identified by qRT-PCR using Rotor-GeneQ (Qiagen, Hilden, Germany) with TransStart Green qPCR SuperMix UDG (Transgene, Beijing, China). The gene-specific primers were designed by primer 6.0. Using two-step method for qPCR, and the genes expression quantity were analyzed by 2^−ΔΔCT method using the A. annua actin sequence as the internal reference gene (Additional file 1: Table S1). We all performed three biologic repetition for each sample. Each 20 μL reaction mixture contained 10 μL of TransStart Green qPCR SuperMix UDG, 1 μL of diluted cDNA, 0.4 μL of each primer (Forward/Reverse primer, 10 μM), 8.2 μL of double distilled water. The qPCR cycling conditions were as follows: 50 °C for 2 min; followed by 40 cycles of 94 °C for 5 s, and 60 °C 30 s in PCR strip tubes [39 (link), 40 (link)].