Uefuji Hirotaka, Tatsumi Yuko, Morimoto Masayuki, Kaothien-Nakayama Pulla, Ogita Shinjiro, Sano Hiroshi
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara, 630-0192, Japan.
Plant Mol Biol. 2005 Sep;59(2):221-7. doi: 10.1007/s11103-005-8520-x.
Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 microg g(-1) fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.
咖啡因（1,3,7 - 三甲基黄嘌呤）在咖啡植物中是由黄苷通过甲基的三次连续转移和一次核糖去除而衍生而来。甲基转移由N - 甲基转移酶催化，即黄苷甲基转移酶（XMT）、7 - 甲基黄嘌呤甲基转移酶（MXMT）和3,7 - 二甲基黄嘌呤甲基转移酶（DXMT）。我们之前从咖啡植物中克隆了编码这些N - 甲基转移酶的三个基因，并在体外重建了咖啡因合成途径的最终序列。在本研究中，我们使用多基因转移方法在烟草植物（烟草）中同时表达这些咖啡基因，并证实所得转基因植物叶片中咖啡因产量成功达到5微克/克鲜重。然后研究了它们对烟草夜蛾（斜纹夜蛾）取食行为的影响，烟草夜蛾会损害多种作物。叶盘选择试验表明，毛虫选择性地取食野生型对照材料，或积极避开转基因材料。结果表明了一种通过在植物中产生咖啡因来赋予植物自我防御能力的新方法。含有咖啡因的第二代转基因作物未来可能会节省劳动力和农业成本，同时减轻农药对环境的负荷。
