Isolation of Dwarf Gene in Echinacea purpurea and Generation of Transgenic Plants Transformed with Dwarf RNA Interference Construct

DWARF gene of Echinacea purpurea was identified by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplication of cDNA end PCR. EpDWARF includes 1,416 bp open reading frame encoding 471 amino acid residues. Phylogenetic analysis reveals that EpDWARF has an high homology with ZeCPD1 (constitutive photomorphogenesis and dwarfism) of Zinnia elegans and AaCYP (cytochrome P450) of Artemisia annua. This suggests that EpDWARF may be a brassinosteriod C-23 hydroxylase catalyzing C-23 hydroxylation of various 22-hydroxylated brassinosteroids (BRs). BR is a class of poly-hydroxylated plant steroidal hormone that influences diverse physiological processes, including embryogenesis, cell elongation, vascular differentiation, fertility, and senescence. DWARFs are key enzymes of BR biosynthesis. Knock-out or mutation of DWARFs has been known to result in dwarf phenotypes and severe defects in plants. RNA interference vector (pFGC5941/EpDWARF) expressing hairpin double-stranded RNA was constructed to induce sequence-specific RNA silencing, resulting in knock-down of EpDWARF. Putative transgenic plants were generated after transformation with recombinant Agrobactium harboring RNA interference vector. The presence of RNA interference construct and the suppression of EpDWARF in transgenic plants were determined by genomic DNA PCR and RT-PCR. In genomic DNA PCR, two transgenic plants had been proved to contain RNA interference construct in genomic DNA. In addition, the content of BR and morphological changes in transgenic plants displaying dwarf phenotypes were analyzed to estimate the activity of EpDWARF. This research was supported by a grant (112019-05-1-SB010) from Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries.