Adal Ayelign M, Sarker Lukman S, Lemke Ashley D, Mahmoud Soheil S
Department of Biology, University of British Columbia, 1177 Research Rd, Kelowna, BC. V1V 1V7, Canada.
Plant Mol Biol. 2017 Apr;93(6):641-657. doi: 10.1007/s11103-017-0588-6. Epub 2017 Mar 3.
A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K and k of 3.69 ± 1.17 µM and 2.01 s respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k /K = 0.56 µMs) than NPP (k /K = 0.044 µMs). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8-72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.
从杂薰衣草中分离出一个茉莉酸甲酯响应性的3-蒈烯合酶（Li3CARS）基因，并在体外对其进行了功能鉴定。薰衣草产生的精油主要由单萜类化合物组成，其中包括具有强效抗菌和杀虫作用的单萜3-蒈烯。在本研究中，我们从杂薰衣草中分离出一个叶片特异性的、茉莉酸甲酯（MeJA）响应性的单萜合酶（Li3CARS）并对其进行了功能鉴定。去除转运肽后的开放阅读框编码一个64.9 kDa的蛋白质，该蛋白在大肠杆菌中表达，并通过镍-亚氨基二乙酸琼脂糖亲和层析进行纯化。重组Li3CARS将香叶基焦磷酸（GPP）转化为主要产物3-蒈烯，其Km和kcat分别为3.69±1.17 μM和2.01 s-1。Li3CARS也以焦磷酸金合欢酯（NPP）作为底物产生多种产物，包括少量的3-蒈烯。Li3CARS产生3-蒈烯的催化效率对于GPP（kcat/Km = 0.56 μM-1s-1）比NPP（kcat/Km = 0.044 μM-1s-1）高十倍以上。Li3CARS利用不同底物（GPP与NPP）产生不同的终产物谱表明，单萜代谢可能部分受底物可用性的控制。与花组织相比，Li3CARS转录本在叶片中高度丰富（16倍）。Li3CARS的转录活性与3-蒈烯的产生相关，并且在MeJA处理后8-72小时上调（1.18至3.8倍）。结果表明，Li3CARS可能在薰衣草中具有防御作用。
