白木香丙二烯氧化物合酶基因的表达分析

发布时间：2018-01-19 09:35

本文关键词： 白木香丙二烯氧化物合酶茉莉酸原核表达表达分析　出处：《药学学报》2017年12期 　论文类型：期刊论文

【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(Aquilaria sinensis)为材料,设计特异引物,克隆了白木香As AOS1基因的c DNA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 AOS1蛋白与甜橙(Citrus sinensis)AOS蛋白同源性较高。构建原核表达载体p ET28a-As AOS1并在大肠杆菌BL21(DE3)菌株中成功表达As AOS1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 AOS1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As AOS1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。
[Abstract]:Jasmonic acid jasmonic acid jasmonic acid (jasmonic) is an important signal molecule in plant defense response. The key enzyme of its biosynthesis pathway is allene oxide synthase. In this study, a specific primer was designed to clone the c DNA sequence of as AOS1 gene from Aquilaria sinensis. Amino acid sequence analysis, prokaryotic expression and purification. Tissue specific analysis, abiotic stress and hormone induced expression analysis showed that the open reading frame (ORF) of as AOS1 gene was 1 575 BP long and encoded 524 amino acids. The molecular weight of its protein is 58.70k Da.As AOS1, which contains the conserved region of cytochrome P450. Phylogenetic tree shows that as AOS1 protein and sweet orange (. Construction of prokaryotic expression vector p ET28a-As AOS1 and expression in Escherichia coli BL21DDE3). The recombinant protein of as AOS1 was successfully expressed in the strain. Soluble as AOS1 recombinant protein was purified by Ni2 affinity chromatography. The results of fluorescence quantitative PCR showed that as AOS1 gene expressed the highest in stem, followed by root, lowest in leaf and salt. Low temperature and heavy metal stress could induce the expression of as AOS1 gene in the callus, and the expression reached the highest level at 12 h. After induction with methyl jasmonate, salicylic acid and abscisic acid, the expression of as AOS1 gene in callus increased significantly. However, drought stress and gibberellin treatment had little effect on the expression of as AOS1 gene. This study laid a foundation for further study on the role of jasmonic acid pathway in the aroma formation mechanism and plant defense response.
【作者单位】：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;
【基金】：国家自然科学基金青年基金资助项目(81503182)
【分类号】：S567.19
【正文快照】： (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 MateriaMedica,China Academy of Chinese Medical Sciences,Beijing 100700,China)同

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