Surrogate splicing for functional analysis of sesquiterpene synthase genes

S Wu, MA Schoenbeck, BT Greenhagen… - Plant …, 2005 - academic.oup.com
S Wu, MA Schoenbeck, BT Greenhagen, S Takahashi, S Lee, RM Coates, J Chappell
Plant physiology, 2005academic.oup.com
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 …
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.
Oxford University Press
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