Wu Shuiqin, Schoenbeck Mark A, Greenhagen Bryan T, Takahashi Shunji, Lee Sungbeom, Coates Robert M, Chappell Joseph
Plant Physiology, Biochemistry and Molecular Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546-0312.
Plant Physiol. 2005 Jul;138(3):1322-33. doi: 10.1104/pp.105.059386. Epub 2005 Jun 17.
A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing beta-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for alpha-barbatene, thujopsene, and beta-chamigrene biosynthesis.
本文描述了一种从含有内含子的预测萜烯合酶基因中回收全长cDNA的方法。该方法利用农杆菌介导的瞬时表达与逆转录-多脱氧核糖核苷酸链反应分析相结合，以促进加工转录本的表达克隆。随后在合适的原核宿主中表达无内含子的cDNA，可对编码的基因产物进行直接功能测试。通过检测农杆菌渗入矮牵牛（Petunia hybrida）叶片中的含内含子β-葡萄糖醛酸酶基因的表达对该方法进行了优化，并通过确定两种先前未表征的萜烯合酶的功能证明了其效用。一个含有六个预测内含子的烟草（Nicotiana tabacum）萜烯合酶样基因被鉴定为具有5-表-马兜铃烯合酶活性，而一个先前注释为萜烯合酶的拟南芥（Arabidopsis thaliana）基因被证明具有用于α-巴巴烯、土荆芥烯和β-菖蒲二烯生物合成的新型倍半萜烯合酶活性。
