董先娟, 李铁铮, 冯莹莹, 王晓晖, 高博闻. 白木香丙二烯氧化物环化酶基因(AsAOC1)的克隆与表达分析J. 药学学报, 2018,53(3): 467-475. doi: 10.16438/j.0513-4870.2017-0988
引用本文: 董先娟, 李铁铮, 冯莹莹, 王晓晖, 高博闻. 白木香丙二烯氧化物环化酶基因(AsAOC1)的克隆与表达分析J. 药学学报, 2018,53(3): 467-475. doi: 10.16438/j.0513-4870.2017-0988
DONG Xian-juan, LI Tie-zheng, FENG Ying-ying, WANG Xiao-hui, GAO Bo-wen. Cloning and expression analysis of allene oxide cyclase gene from Aquilaria sinensisJ. Acta Pharmaceutica Sinica, 2018,53(3): 467-475. doi: 10.16438/j.0513-4870.2017-0988
Citation: DONG Xian-juan, LI Tie-zheng, FENG Ying-ying, WANG Xiao-hui, GAO Bo-wen. Cloning and expression analysis of allene oxide cyclase gene from Aquilaria sinensisJ. Acta Pharmaceutica Sinica, 2018,53(3): 467-475. doi: 10.16438/j.0513-4870.2017-0988

白木香丙二烯氧化物环化酶基因(AsAOC1)的克隆与表达分析

Cloning and expression analysis of allene oxide cyclase gene from Aquilaria sinensis

  • 摘要: 丙二烯氧化物环化酶(allene oxide cyclase,AOC)是茉莉酸生物合成途径中的一个关键酶,在植物防御反应中发挥重要作用。本研究根据白木香转录组高通量测序结果设计引物扩增,利用RT-PCR技术从白木香中首次克隆得到1个AOC基因,命名为AsAOC1,并进行生物学信息分析、原核表达和纯化、组织特异性分析及非生物胁迫和激素诱导表达分析。白木香AsAOC1基因的开放阅读框(ORF)长753 bp,编码251个氨基酸,其蛋白分子质量是27.46 kD。生物学信息分析表明AsAOC1蛋白包含1个保守的allene_ox_cyc结构域,系统进化树显示AsAOC1蛋白与川桑(Morus notabilis) AOC蛋白具有较高的同源性。构建原核表达载体pET28a-AsAOC1并在大肠杆菌BL21(DE3)菌株中成功表达AsAOC1重组蛋白,利用Ni2+亲和色谱纯化得到可溶性AsAOC1重组蛋白。实时荧光定量PCR检测结果表明AsAOC1基因在茎中表达量最高,根与茎尖次之,叶中表达量最低。盐、干旱、低温以及重金属胁迫均能够诱导白木香愈伤组织中AsAOC1基因表达;茉莉酸甲酯、水杨酸、赤霉素和脱落酸诱导后,愈伤组织中AsAOC1基因的表达量明显上升。本研究为进一步研究茉莉酸途径在白木香结香机制和植物防御反应中作用奠定基础。

     

    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.

     

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