RESUMEN
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.
RESUMEN
Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo- (10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.