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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes^1,[w]

Plant Physiology, Biochemistry and Molecular Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546–0312 (S.W., M.A.S., B.T.G., S.T., S.L., J.C.); and Department of Chemistry, University of Illinois, Urbana, Illinois 61801 (R.M.C.)

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 -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 -barbatene, thujopsene, and -chamigrene biosynthesis.

² These authors contributed equally to the paper.

³ Present address: Biology Department, University of Nebraska-Omaha, Omaha, NE, 68182.

⁴ Present address: Allylix Inc., A165 ASTeCC, University of Kentucky, Lexington, KY 40546–0286.

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059386.

^* Corresponding author; e-mail chappell{at}uky.edu; fax 859–257–7125.

Received January 6, 2005; returned for revision February 23, 2005; accepted February 24, 2005.