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  本文關(guān)鍵詞： 白木香 丙二烯氧化物合酶 茉莉酸 原核表達 表達分析　出處：《藥學(xué)學(xué)報》2017年12期 　論文類型：期刊論文

【摘要】：茉莉酸(jasmonic acid,JA)為植物防御反應(yīng)的重要信號分子,其生物合成途徑的關(guān)鍵酶是丙二烯氧化物合成酶(allene oxide synthase,AOS)。本研究以白木香(Aquilaria sinensis)為材料,設(shè)計特異引物,克隆了白木香As AOS1基因的c DNA序列,并進行氨基酸序列分析、原核表達和純化、組織特異性分析及非生物脅迫和激素誘導(dǎo)表達分析。白木香As AOS1基因的開放閱讀框(ORF)長1 575 bp,編碼524個氨基酸,其蛋白分子質(zhì)量是58.70 k Da。As AOS1蛋白包含細胞色素P450的保守區(qū)域。系統(tǒng)進化樹顯示As AOS1蛋白與甜橙(Citrus sinensis)AOS蛋白同源性較高。構(gòu)建原核表達載體p ET28a-As AOS1并在大腸桿菌BL21(DE3)菌株中成功表達As AOS1重組蛋白,利用Ni2+親和色譜純化得到可溶性As AOS1重組蛋白。熒光定量PCR結(jié)果顯示As AOS1基因在莖中表達最高,根次之,葉中表達最低。鹽、低溫和重金屬脅迫能夠誘導(dǎo)白木香愈傷組織中As AOS1基因表達,誘導(dǎo)12 h表達量達到最高;激素茉莉酸甲酯、水楊酸和脫落酸誘導(dǎo)后,愈傷組織中As AOS1基因的表達量明顯上升,而干旱脅迫和赤霉素處理對本As AOS1基因的表達量影響不大。本研究為進一步研究茉莉酸途徑在白木香結(jié)香機制和植物防御反應(yīng)中作用奠定基礎(chǔ)。
[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.
【作者單位】： 北京中醫(yī)藥大學(xué)中藥學(xué)院中藥現(xiàn)代研究中心;中國中醫(yī)科學(xué)院中藥研究所;
【基金】：國家自然科學(xué)基金青年基金資助項目(81503182)
【分類號】：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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