白木香丙二烯氧化物合酶基因的表达分析.docxVIP

白木香丙二烯氧化物合酶基因的表达分 冯莹莹张钟秀 董先娟 刘晓闫雅如 王金铃 王晓 北京中医药大学中药学院中药现代研究中心 中国中 医科学院中药研究所 摘要： 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(Aqu订aria sinensis)为材料，设计特异引物，克隆了白木香As AOS1 基因的cDNA序列，并进行氨基酸序列分析、原核表达和纯化、组织特界性分析 及非牛物胁迫和激素诱导表达分析。白木香As AOS1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 k Da。As AOS1蛋白包 含细胞色素P450的保守区域。系统进化树显示As A0S1蛋白与甜橙(Citrus sinensis) AOS蛋口同源性较高。构建原核表达载体p ET28a~As A0S1并在大肠 杆菌BL21 (DE3)菌株中成功表达As A0S1重组蛋白，利用Ni2+亲和色谱纯化得 到可溶性As AOS1重组蛋白。荧光定量PCR结果显示As AOS1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 As A0S1基因表达，诱导12 h表达量达到最高;激素茉莉酸甲酯、水杨酸和脫落 酸诱导后，愈伤组织中As A0S1基因的表达量明显上升，而干旱胁迫和赤霉素处 理对本As AOS1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。 关键词： 口木香;丙二烯氧化物合酶;茉莉酸;原核表达;表达分析; 作者简介：王晓晖 Te 1:86-10E-ma订：wangxhui2014@163. com; 作者简介：史社坡 Tel:86-10 E-ma订:shishepo@163. com 收稿日期：2017-05-14 基金：国家自然科学基金青年基金资助项目 Expression analysis of allene oxide synthase gene from Aquilaria sinensis FENG Ying-ying ZHANG Zhong-xiu DONG Xian-juan LIU Xiao YAN Ya-ru WANG Jin-ling WANG Xiao-hui SHI She-po Modern Research Center for Troditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine; Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences; Abstract： Jasmonic acid (JA) is an important signal molecule invoIved in plant resistanee, and allene oxide synthase (AOS) is a key enzyme in the biosynthesis of jasmonates. In this study, a full-length c DNA of As AOS1 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 freime (ORF) of As AOS1 gene was 1 575 bp, encoding a protein of 524 amino acid residues, with a predicted molecular mass of 58.70 k Da. As AOS1 protein possessed the conserved sequences of cytochrome P450 (CYP450) . The phylogenetic a