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

ABSTRACT

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.