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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。
JA (jasmonic acid) is an important signaling molecule in plant defense response. The key enzyme in biosynthesis pathway is allene oxide synthase (AOS). In this study, we designed a specific primer using Aquilaria sinensis as material, cloned the c DNA sequence of AsAOS1 gene and analyzed its amino acid sequence, prokaryotic expression and purification, tissue-specific analysis, abiotic stress and hormone induction Expression analysis. The open reading frame (ORF) of AsAOS1 gene is 1 575 bp in length and encodes a protein of 524 amino acids with an estimated molecular mass of 58.70 kDa. As AOS1 protein contains a conserved region of cytochrome P450. The phylogenetic tree shows that As AOS1 protein has high homology with AOS protein of Citrus sinensis. The prokaryotic expression vector p ET28a-As AOS1 was constructed and the As AOS1 recombinant protein was successfully expressed in E. coli BL21 (DE3). The soluble As AOS1 recombinant protein was purified by Ni2 + affinity chromatography. Fluorescence quantitative PCR results showed that As AOS1 gene expressed highest in the stem, followed by roots, and lowest in the leaves. Salinity, low temperature and heavy metal stress induced the expression of As AOS1 gene in the calluses of Baizhuyamang, and reached the highest level at 12 h after induction. After induced by methyl jasmonate, salicylic acid and abscisic acid, the expression of As AOS1 gene While the expression of As AOS1 gene was not significantly affected by drought stress and gibberellin treatment. This study lays the foundation for further study of the role of jasmonic acid pathway in the mechanism of plantation of white wood and the plant defense response.