Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes

Author:

Wu Shuiqin1,Schoenbeck Mark A.1,Greenhagen Bryan T.1,Takahashi Shunji1,Lee Sungbeom1,Coates Robert M.1,Chappell Joseph1

Affiliation:

1. 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.)

Abstract

Abstract 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 β-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 α-barbatene, thujopsene, and β-chamigrene biosynthesis.

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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