Cloning and expression analysis of gibberellin 3-oxidase gene in Dendrobium officinale / 中草药

ABSTRACT

Objective: To clone the gibberellin 3-oxidase gene DoGA3ox from an important and endangered medicinal plant Dendrobium officinale (Orchidaceae), followed by bioinformatic and expression analysis. Methods: RACE and RT-PCR were used to isolate the full-length gene. The physicochemical properties, conserved domains, and subcellular localization of DoGA3ox protein were determined using a series of bioinformatic tools. The phylogenetic analyses were performed using DNASTAR 7.0 and MEGA 6.0 software. Real time quantitative PCR was employed for gene expression analysis. Results: The full-length cDNA of DoGA3ox (GenBank registration number KT597694) was 1 318 bp in size, and encoded a 353-amino acid peptide chain with a molecular weight of 39052.5 and an isoelectric point (pI) of 6.21; The deduced DoGA3ox protein without transmembrane or signal peptide residues, contained the gibberellin 3-oxidases conserved domains that DIOX_N (40-130) and 2OG-FeII_Oxy (197-299). DoGA3ox had 51%-56% similarity with GA3ox proteins from various plants, and was closely related to the monocot Allium fistulosum, Elaeis guineensis, and Phoenix dactylifera. The relative transcript levels of DoGA3ox were increased at stage 1, then decreased (stage 2), and increased again (stage 3) during D. officinale symbiotic/asymbiotic seed germination, the fold change to ungerminated seeds was 13.44 (symbiotic)/5.21 (asymbiotic), 7.28/2.32 and 9.40/6.21, respectively. Moreover, its transcript level was higher in symbiotic germination than that in the asymbiotic status. Conclusion: The full-length DoGA3ox gene is cloned for the first time, and its expression in different stages during seed germination indicates that DoGA3ox gene plays a crucial role in the regulation of seed germination in D. officinale.