Cloning and expression analysis of gene DoSRK2E in Dendrobium officinale / 中草药

ABSTRACT

Objective To clone the SnRK2 gene in Dendrobium officinale and investigate its characteristics and expression pattern. Methods RT-PCR and RACE techniques were used to clone the full-length cDNA of DoSRK2E, with the aids of a series of online bioinformatic software, characteristics including molecular mass, isoelectric point, conserved domain, transmembrane structure, signal peptide, and subcellular localization of the deduced protein were analyzed. Besides, the sequence of the deduced protein was aligned with those of other plant SnRK2 by DNASTAR, and phylogenetic relationships were reconstructed utilizing MEGA. Finally, tissue specific expression pattern of DoSRK2E was tested by quantitative real-time PCR (qRT-PCR). Results The full-length cDNA of DoSRK2E (GenBank accession API65110) is 1 795 bp with a 1 086 bp complete open reading frame (ORF). The predicted molecular mass and isoelectric point of the deduced protein DoSRK2E were 40 850 and 4.80, respectively. No signal peptide nor transmembrane region were detected, this protein contains one protein kinase domain, one ATP binding site, and one Ser/Thr active site, which was predicted most likely to be located in the endoplasmic reticulum membrane. DoSRK2E protein showed high similarity with those from other plant SnRK2, and its phylogenetic location was in Group III of SnRK2 subfamily, and phylogenetically closest to AtSnRK2.6 from Arabidopsis. In addition, qPCR analysis revealed that DoSRK2E showed the highest expression level in root, followed by stem and leaf. Conclusion A SnRK2 family gene DoSRK2E was cloned from the rare and endangered medicinal plant D. officinale for the first time. The Characteristics and expression pattern of this gene were analyzed. This study will provide a basis for further exploration of the regulation mechanisms of DoSRK2E in D. officinale under stress conditions.