Gene cloning, subcellular localization and expression analysis of the AsERF1 gene from Aquilaria sinensis / 药学学报

Ethylene-response factors, which are a subfamily of the AP2/ERF family, play an important role in ethylene signal transduction, plant growth and plant resistant. In this study, a full-length cDNA of the AsERF1 gene was cloned from Aquilaria sinensis. Sequence analysis, prokaryotic expression and purification, subcellular localization, tissue-specific analysis and expression analysis under different abiotic stresses was performed. The open reading frame (ORF) of the AsERF1 gene was 691 bp, encoding a protein of 229 amino acids with a predicted molecular mass of 25.36 kD. The AsERF1 protein contained the conserved AP2 sequence of ERF protein. A phylogenetic analysis indicated that the AsERF1 protein showed greatest sequence similarity with ERF2 from Populus trichocarpa. The recombinant AsERF1 protein was expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsERF1 and the recombinant AsERF1 protein was purified. Agrobacterium-mediated protein expression experiments demonstrated that AsERF1 mainly localized to the nucleus. Expression analysis indicated that AsERF1 was primarily observed in leaves. The AsERF1 expression level was induced by salt, drought, low temperature and CdCl2 treatment, while the abundance of AsERF1 was most significantly induced by drought stress. These results provide valuable insights into the role of AsERF1 in plant defense and the mechanism of agarwood formation.

Cloning and expression analysis of allene oxide cyclase gene from Aquilaria sinensis / 药学学报

Allene oxide cyclase (AOC), a key enzyme in biosynthesis of jasmonic acid, plays an essential role in the plant defense system. In present study, a full length cDNA of AsAOC gene was cloned by the reverse transcription PCR from Aquilaria sinensis calli. Meanwhile, the bioinformatics, prokaryotic expression, purification, tissue-specific expression analysis, and expression analysis under different abiotic stresses and hormone treatments were performed. The open reading frame (ORF) of AsAOC1 gene was 753 bp, encoding a protein of 251 amino acids with a calculated molecular mass (MW) of 27.46 kD. Bioinformatic analysis showed that AsAOC1 protein contains a conserved allene_ox_cyc domain in C-terminus. The phylogenetic analysis indicated that AsAOC1 protein had the highest level of homology with the AOC protein from Morus notabilis. The recombinant AsAOC1 protein was successfully expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsAOC1 and was purified by Ni2+ affinity chromatography. Expression analysis in different tissues indicated that AsAOC1 was primarily observed in stems, and then stem tips and roots, following by leaves. The transcript level of AsAOC1 was induced by various abiotic stresses including salt, drought, cold, and heavy metal stress. Furthermore, AsAOC1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA), gibberellin (GA3), and abscisic acid (ABA) treatments. These results provide valuable insights into the role of JA in the mechanism of agarwood formation and plant defense system.