Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum
Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum
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DOI：
10.1021/np960344w
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发表时间：
1996-10-01
影响因子：
5.1
通讯作者：
Heinstein, P
中科院分区：
文献类型：
--
作者：
Chen, XY;Wang, MS;Heinstein, P
一株亚洲棉的筛选。南京文库的构建成功地鉴定和克隆了第二个(+)-β-钙质烯合成酶。以法尼基二磷酸环化酶保守序列为基础，以基因组DNA为模板，通过聚合酶链式反应(PCR)制备筛选探针。这第二个克隆编码的蛋白质与最近描述的亚洲棉(G.arboreum)中的(+)-β-杜松烯合成酶CAD_1-C_1和C_(14)有80%的同源性，并与其他已知的单萜、倍半烯和二萜合成酶保持着显著的同源性。与从棉花细胞培养的CAD1-C1(+)-Delta-Cadinene合成酶的情况一样，棉花细胞悬浮培养物用来自植物病原真菌黄萎病菌的部分纯化的激发子制剂处理棉花细胞，可以诱导第二个CAD1-AmRNA的合成。逆转录-聚合酶链式反应(RT-PCR)检测CAD1-AmRNA的表达，发现激发子加入后6h，CAD1-AmRNA的表达最高，为对照组的8倍。这第二个基因的异源表达产生了一个KD蛋白，该蛋白催化法尼基二磷酸环化为(+)-β-杜松烯，这与CAD-C1产生的相同产物。CAD1A和CAD1C的稳态动力学参数相似，30℃时Km值为7 mM法尼基二磷酸，k(CAT)为0.039 S(-1)，但CAD1A活性的最适pH和镁离子浓度显著高于CAD1C。
Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template. This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G. arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases. As in the case, of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae. Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor. Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD-C1. The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a K-m of 7 mM farnesyl diphosphate and k(cat) of 0.039 s(-1) at 30 degrees C. However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C.