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Published on June 17, 2005; 10.1104/pp.105.059386

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Received January 6, 2005
Returned for revision February 23, 2005
Accepted February 24, 2005

Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes

Shuiqin Wu , Mark A. Schoenbeck , Bryan T. Greenhagen , Shunji Takahashi , Sungbeom Lee , Robert M. Coates , and Joseph Chappell *

Plant Physiology, Biochemistry and Molecular Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546-0312
Department of Chemistry, University of Illinois, Urbana, Illinois 61801

* Corresponding author; email: chappell{at}uky.edu.

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.






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