Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 5.53

CHARACTERIZING THE ACYL CARRIER PROTEIN GENE IN OLIVE

L. BALDONI, C. RICCIOLINI, A. PORCEDDU, E. VARASANO, C. GUERRERO

CNR - Istituto di Genetica Vegetale, Sezione di Perugia

Lipid metabolism in olive has been mainly studied at biochemical level but only few works were addressed to the identification and characterization of genes involved in the process of oil accumulation in the fruits, such as the Stearoyl-ACP Desaturase and the Diacylglicerol Acetyl Transferase (DGAT).

Plants need Acyl Carrier Protein (ACP) for the synthesis of fatty acids in membranes and for lipid accumulation. Numerous isoforms of ACP have been described in different plant species, permitting the fine regulation of fatty acid synthesis. ACP is a small protein (approximately 9,500 Da) which plays an important role as cofactor in all reactions of fatty acid synthesis in the chloroplast transferring new acyl groups to the borning chain up to the production of the first unsaturated fatty acid (oleic acid). ACP is a very asbundant component of the fatty acid synthesis, due to the fact that most of the enzymes require acyl-ACP in their reactions. It has been the first protein of plant lipid metabolism to be purified and the first whose gene was cloned. Numerous ACP genes and cDNA clones have been characterized in plants, like in Brassica, spinach, Cuphea and Arabidopsis.

The role played by ACP in the process of oil synthesis in olive fruits is under study and, in this context, the identification and characterization of the ACP gene were carried out. A cDNA library from developing fruits has been screened with an heterologous probe. From partial cDNA clones the 5’ end of the messanger was obtained by a 5’RACE experiment. The complete 763 bp cDNA clone, coding for a peptide of 134 residues including the signal peptide followed by the mature protein, has been obtained and its sequence shows high homology with that of other plant species. From the sequencing of the RACE product resulted the presence of four different clones almost identical but differing in three nucleotides and a deletion. These differences caused a change in the protein sequence only in two cases. The expression of the ACP gene during olive fruit ripening has been studied by Northern analysis of RNA from the Hojiblanca cultivar. The level of ACP messanger starts to increase when the oil synthesis starts, as it happens for an other important oil synthesis gene in olive, the Stearoyl-ACP Desaturase. The isolation of various genomic clones of ACP in different plants has shown that the corresponding genes present a mosaic conserved structure with four exons interrupted by three introns and the position of these introns is the same in many species. By PCR amplification of the genomic regions corresponding to the different parts of the cDNA clone it was possible to clarify the position and sequence of introns in the olive gene. A fragment from the cDNA clone has been used for Southern analysis in order to establish the number of ACP gene copies present in olive.