Genomic DNA of A. annua was isolated using the CTAB method. A genome walking method, that is, fusion primer and nested integrated PCR (Wang et al., 2011 (link)) was carried out to amplify the promoter of AaDXS2 (pAaDXS2). Primers used to amplify pAaDXS2 are listed in Supplementary Table S1 . The TSSP software was used to determine the transcription start site of AaDXS2 (Solovyev and Shahmuradov, 2003 (link)). The cis-elements in pAaDXS2 were analyzed using PlantCARE website1 and PLACE website2 .
To investigate the expression pattern of AaDXS2 in plants, the pAaDXS2 was cloned into pCAMBIA1391.Z to drive the expression of GUS gene. The pAaDXS2::GUS construct was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into A. thaliana by the floral dip method (Zhang et al., 2006 (link)). Mature leaves and flowers of 45-day-old arabidopsis seedlings as well as siliques from 2-month-old transgenic A. thaliana were used for GUS histochemical staining as described previously (Jefferson et al., 1987 (link)). GUS stained tissues were observed under Olympus SZX16 microscope, and pictures were taken using Olympus DP73 digital camera.
To investigate the expression pattern of AaDXS2 in plants, the pAaDXS2 was cloned into pCAMBIA1391.Z to drive the expression of GUS gene. The pAaDXS2::GUS construct was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into A. thaliana by the floral dip method (Zhang et al., 2006 (link)). Mature leaves and flowers of 45-day-old arabidopsis seedlings as well as siliques from 2-month-old transgenic A. thaliana were used for GUS histochemical staining as described previously (Jefferson et al., 1987 (link)). GUS stained tissues were observed under Olympus SZX16 microscope, and pictures were taken using Olympus DP73 digital camera.
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