Colby S M, Crock J, Dowdle-Rizzo B, Lemaux P G, Croteau R
U.S. Department of Agriculture/University of California, Berkeley, Plant Gene Expression Center and Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2216-21. doi: 10.1073/pnas.95.5.2216.
Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).
通过气相色谱-质谱联用（GC-MS）和核磁共振（NMR）分析发现，番茄品种VFNT Cherry叶油中含量最丰富的倍半萜是杜松烯C，杜松烯A、愈创木-6,9-二烯、杜松烯B、β-石竹烯、α-葎草烯和杜松烯D的含量较少。叶中可溶性酶制剂催化了[1-³H]法呢基二磷酸的二价金属离子依赖性环化反应，生成这些相同的倍半萜烯烃，这是通过放射性气相色谱法测定的。为了获得杜松烯合酶cDNA，基于相关萜类环化酶的保守氨基酸序列构建了一组简并引物。利用从富含叶表皮的mRNA制备的cDNA，这些引物扩增出一个767 bp的片段，该片段用作杂交探针来筛选cDNA文库。通过使用标记的香叶基、法呢基和香叶基香叶基二磷酸作为底物，对31个克隆进行了在大肠杆菌中萜类环化酶活性的功能表达评估。9个cDNA分离物表达了倍半萜合酶活性，产物的GC-MS分析鉴定出杜松烯C以及少量的杜松烯A、B和D。所表达的蛋白质均对香叶基香叶基二磷酸无活性；然而，一种截短的蛋白质将香叶基二磷酸转化为单萜柠檬烯。cDNA插入片段指定了一个由548个氨基酸组成的推导多肽（Mr = 64,114），与其他植物倍半萜环化酶的序列比较表明，杜松烯C合酶与棉花δ-荜澄茄烯合酶最为相似（同一性为50%）。
