Gene family for an elicitor-induced sesquiterpene cyclase in tobacco

P. J. Facchini, J. Chappell

Research output: Contribution to journalArticlepeer-review

212 Scopus citations

Abstract

The initial step in the conversion of the isoprenoid intermediate farnesyl diphosphate to the sesquiterpenoid phytoalexin capsidiol in elicitor-treated tobacco tissues is catalyzed by an inducible sesquiterpene cyclase [5-epi- aristolochene synthase (EAS)]. Two independent cDNA clones (cEAS1 and cEAS2) encoding EAS were isolated from an elicitor-induced tobacco cDNA library by differential hybridization and subsequently were characterized by hybrid selection-in vitro translation. Insertion of cEAS1, a partial cDNA clone encoding 175 C-terminal amino acids, into an Escherichia coli expression vector resulted in accumulation of a fusion protein immunodetectable with EAS-specific polyclonal antibodies. The cDNA clones were used to isolate two full-length EAS genes that mapped 5 kilobases (kb) apart on one 15-kb genomic clone. The nucleotide sequences of the structural gene components were identical from 388 base pairs (bp) upstream of the transcription initiation site to 40 bp downstream of the translation termination codon, suggesting a relatively recent duplication event. The genes consist of 1479-bp open reading frames, each containing five introns and specifying 56,828-Da proteins. The N-terminal amino acid sequence deduced from the genomic clones was identical to the first 16 amino acids of the EAS protein identifiable by Edman degradation. RNA blot hybridization with cEAS1 demonstrated a mRNA induction time course consistent with the induction of the EAS mRNA translational activity with maximum levels 4-6 h after elicitation. EAS mRNA was not detected in control cells. DNA blot-hybridization analysis of genomic DNA revealed a copy number of ≃12-15 for EAS-like genes in the tetraploid tobacco genome. The conservation of a putative allelic prenyl diphosphate binding motif is also discussed.

Original languageEnglish
Pages (from-to)11088-11092
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number22
DOIs
StatePublished - 1992

Keywords

  • heterologous gene expression
  • isoprenoids
  • terpene cyclases

ASJC Scopus subject areas

  • General

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