Surrogate splicing for functional analysis of sesquiterpene synthase genes.
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.
Wu
Shuiqin
S
Plant Physiology, Biochemistry and Molecular Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546-0312.
Schoenbeck
Mark A
MA
Greenhagen
Bryan T
BT
Takahashi
Shunji
S
Lee
Sungbeom
S
Coates
Robert M
RM
Chappell
Joseph
J
D017398:Alternative Splicing; D019755:Carbon-Carbon Lyases / Q000235:genetics*; D018076:DNA, Complementary; D017930:Genes, Reporter; D005966:Glucuronidase / Q000378:metabolism; D007438:Introns; D032306:Petunia / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified; D015183:Restriction Mapping; D012231:Rhizobium / Q000235:genetics; D014158:Transcription, Genetic
D018076:DNA, Complementary; D005966:Glucuronidase; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase