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Cloning and functional expression of an (E,E)-a-farnesene synthase cDNA from apple fruit.

Pechous S.W., Whitaker B.D.

5th International Postharvest Symposium . Volume of Abstract . Verona, Italy 6-11 June 2004, p.64

2004

บทคัดย่อ

Cloning and functional expression of an (E,E)-a-farnesene synthase cDNA from apple fruit.  Increased production of terpenes and many other volatiles occurs with ripening in apple fruit.Ethylene plays a key role in induction of volatile synthesis, but the mechanism is not understood.Using a degenerate primer base o­n a short conserved sequence shared by several sequiterpene synthases, RT-PCR with UNA isolated from peel tissue of ‘Law Uome’apples yielded and –800-bp gene fragment.This was used to screen a cKNA libraty generated from peel tissue mRNA.A full-length terpene synthase (TS) cKNA 1931 nucleotides long was isolated.The 1728-bp open reading frame encodes a protein 576 amino acids long with a molecular mass of 66 kDa.

Sequence anlysis of the apple TS showed it to be most similar to several monoterpene synthases (MTSs).The TS inclueds an RR(X8)W motif near the N-terminus that is common among MTSs but it lacks the plastid transit peptide seqrence associated with MTS genes.Expression of the TS gene in bacteria gave epitope-tagged and untagged proteins estimated at ~68 and ~66 kDa, respectively.In sesquitepene synthase assays with farnesy diphosphate as substrate, the untagged bacterially-expressed TS gene product synthesized (E,E)-a-farnesene almost exclusively.In monoterpene synthase assays including geranyl diphosphate, the untagged TS produced o­nly (E)-b-ocimene, albeit at much reduced levels.This is the first report of an (E,E)-a-farnesene synthase gene (AFS1; Genbank accession number AY182241) from a flowering plant.RNA gel blots showed that AFS1 transcript increased fourfold in peel tissue of apple fruit during the first 4 weeks of storage at 0.5 °C.In contrast, when fruit were treated at harvest with 1-methylcyclopropene, a blocker of ethylene action, AFS1 mRNA declined sharply over the initial 4 weeks of cold storage, and was nearly undetectable by 8 weeks.