药学学报, 2017, 52(12): 1962-1969
冯莹莹, 张钟秀, 董先娟, 刘晓, 闫雅如, 王金铃, 王晓晖, 史社坡. 白木香丙二烯氧化物合酶基因的表达分析[J]. 药学学报, 2017, 52(12): 1962-1969.
FENG Ying-ying, ZHANG Zhong-xiu, DONG Xian-juan, LIU Xiao, YAN Ya-ru, WANG Jin-ling, WANG Xiao-hui, SHI She-po. Expression analysis of allene oxide synthase gene from Aquilaria sinensis[J]. Acta Pharmaceutica Sinica, 2017, 52(12): 1962-1969.

冯莹莹1, 张钟秀2, 董先娟1, 刘晓1, 闫雅如1, 王金铃1, 王晓晖1, 史社坡1
1. 北京中医药大学中药学院中药现代研究中心, 北京 100029;
2. 中国中医科学院中药研究所, 北京 100700
茉莉酸(jasmonic acid,JA)为植物防御反应的重要信号分子,其生物合成途径的关键酶是丙二烯氧化物合成酶(allene oxide synthase,AOS)。本研究以白木香(Aquilaria sinensis)为材料,设计特异引物,克隆了白木香AsAOS1基因的cDNA序列,并进行氨基酸序列分析、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AsAOS1基因的开放阅读框(ORF)长1 575 bp,编码524个氨基酸,其蛋白分子质量是58.70 kDa。AsAOS1蛋白包含细胞色素P450的保守区域。系统进化树显示AsAOS1蛋白与甜橙(Citrus sinensis)AOS蛋白同源性较高。构建原核表达载体pET28a-AsAOS1并在大肠杆菌BL21(DE3)菌株中成功表达AsAOS1重组蛋白,利用Ni2+亲和色谱纯化得到可溶性AsAOS1重组蛋白。荧光定量PCR结果显示AsAOS1基因在茎中表达最高,根次之,叶中表达最低。盐、低温和重金属胁迫能够诱导白木香愈伤组织中AsAOS1基因表达,诱导12 h表达量达到最高;激素茉莉酸甲酯、水杨酸和脱落酸诱导后,愈伤组织中AsAOS1基因的表达量明显上升,而干旱胁迫和赤霉素处理对本AsAOS1基因的表达量影响不大。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。
Expression analysis of allene oxide synthase gene from Aquilaria sinensis
FENG Ying-ying1, ZHANG Zhong-xiu2, DONG Xian-juan1, LIU Xiao1, YAN Ya-ru1, WANG Jin-ling1, WANG Xiao-hui1, SHI She-po1
1. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China;
2. Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
Jasmonic acid (JA) is an important signal molecule involved in plant resistance, and allene oxide synthase (AOS) is a key enzyme in the biosynthesis of jasmonates. In this study, a full-length cDNA of AsAOS1 gene was cloned from Aquilaria sinensis. Meanwhile, the sequence analysis, 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 AsAOS1 gene was 1 575 bp, encoding a protein of 524 amino acid residues, with a predicted molecular mass of 58.70 kDa. AsAOS1 protein possessed the conserved sequences of cytochrome P450 (CYP450). The phylogenetic analysis indicated that AsAOS1 protein had the highest level of homology with AOS protein of Citrus sinensis. The recombinant AsAOS1 protein was successfully expressed in Escherichia coli BL21(DE3) cells using the prokaryotic expression vector pET28a-AsAOS1 and the recombinant AsAOS1 was purified by Ni2+ affinity chromatography. Expression analysis results in different tissues showed that AsAOS1 was primarily observed in stems, and then roots, followed by leaves. AsAOS1 transcript level was significantly induced after 12 h treatment of NaCl, cold temperature and CdCl2. Furthermore, AsAOS1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. However, mannitol and gibberellin (GA3) treatments had little influence on the expression level of AsAOS1. These results provides valuable insights into the role of JA in the mechanism of agarwood formation and plant resistance.
Key words:   
收稿日期: 2017-05-14
DOI: 10.16438/j.0513-4870.2017-0568
基金项目: 国家自然科学基金青年基金资助项目(81503182).
通讯作者: 王晓晖,Tel:86-10-64286180,E-mail:wangxhui2014@163.com;史社坡,Tel:86-10-64286350,E-mail:shishepo@163.com
Email: wangxhui2014@163.com;shishepo@163.com
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