Surrogate splicing for functional analysis of sesquiterpene synthase genes

Plant Physiol. 2005 Jul;138(3):1322-33. doi: 10.1104/pp.105.059386. Epub 2005 Jun 17.

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 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alternative Splicing*
  • Carbon-Carbon Lyases / genetics*
  • DNA, Complementary
  • Genes, Reporter
  • Glucuronidase / metabolism
  • Introns
  • Petunia / enzymology*
  • Petunia / genetics
  • Plants, Genetically Modified
  • Restriction Mapping
  • Rhizobium / genetics
  • Transcription, Genetic

Substances

  • DNA, Complementary
  • Glucuronidase
  • Carbon-Carbon Lyases
  • trichodiene synthetase