Project Number: 1275-43000-009-01
Project Type: Reimbursable

Start Date: Jan 01, 2000
End Date: Dec 31, 2004

Objective:
To clone the gene from apple peel tissue that encodes alpha-farnesene synthase and express the cDNA in transformed E. coli or yeast cell lines to obtain functional, purified enzyme for biochemical studies. To determine whether in vivo oxidation of alpha-farnesene in apple peel is enzymatic, and if so, to isolate and purify the oxidase enzyme, and subsequently clone the gene that encodes the enzyme.

Approach:
A cDNA expression library will be generated using mRNA isolated from peel tissue of scald-susceptible apples stored for 1 month at 0C in air. The library will be probed with the cDNA encoding beta-farnesene synthase from mint and/or oligonucleotides based on the amino acid sequence of beta-farnesene synthase from pine to identify the cDNA for alpha-farnesene synthase from apple fruit. The isolated alpha-farnesene cDNA will then be cloned into a vector for transformation of E. coli or yeast cells. Isotopically-labeled alpha-farnesene will be used to assay for enzymatic oxidation to conjugated trienols in peel tissue plugs and cell extracts from scald-susceptible apples. Reaction products will be analyzed by HPLC-UV and GC-MS. If farnesene oxidase activity is found, the enzyme will be isolated, and purified by column chromatography and gel electrophoresis, and partially sequenced. BSL-1 Exempt Recertified 9/7/01.

Project Team

Whitaker, Bruce

Project Annual Reports

FY 2003   FY 2002   FY 2001   FY 2000

Related National Programs

Quality and Utilization of Agricultural Products (306)