Total plant rna extract kit

Quantitative real-time PCR was performed to analyze genes’ expression in this study. Total RNA of root, stem, leaf and flower was extracted using the total plant RNA Extract Kit (Tiangen, China), and then reversely transcribed into cDNA using Fast King RT Kit (Tiangen, China). Subsequently, the cDNA was used as the template for detecting the expression levels of AabHLH112 and sesquiterpene synthase genes AaCPS, AaECS, and AaBFS by qPCR experiment. The qPCR amplification conditions were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 55°C for 20 s, and 72°C for 20 s. β-actin of A. annua was used as the reference gene in this study (Ma et al., 2018 (link)), and the relative expression levels were calculated using the 2^–△△Ct method (Livak and Schmittgen, 2001 (link)). In addition, the expression levels of sesquiterpenes-related genes were detected in AabHLH112-overexpressing and RNAi-AabHLH112 transgenic A. annua using the above method. The primer sequences in qPCR experiment were listed in Supplementary Table 1.

To detect the transgenes, genomic DNA was extracted from the A. annua plants using our previously reported methods (Zhang et al., 2015 (link)). The primers used are listed in Supplementary Table S1. Real-time quantitative PCR (RT-qPCR) analysis was done following previously reported methods (Zhang et al., 2015 (link)). Roots, stems, leaves, and flower buds were collected from 5-month-old plants and immediately frozen in liquid nitrogen. For the plants treated with ABA, NaCl, and drought (and their respective controls) only the leaves were collected. To analyse the expression levels of AaAPK1 and the artemisinin biosynthesis genes, leaves from 2-month-old transgenic and wild-type plants were harvested. The total RNA exacted from these samples was prepared as described above using the total plant RNA Extract Kit (Tiangen, Beijing, China). The relative expression levels of genes were normalized to the expression of A. annua ACTIN. Real-time PCR was performed in a Bio-Rad MJ thermocycler IQ5 (Bio-Rad, Hercules, USA). Amplification was carried out with the Brilliant SYBR Green QPCR MasterMix (Takara, Japan), according to the manufacturer’s instructions. The thermal profile for SYBR Green real-time PCR was 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min.