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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 1.23

2-DE ANALYSIS OF PROTEOME IN VITIS VINIFERA TISSUES

PERUGINI I.*, SCHUBERT A.**

*) Dipartimento Colture Arboree, Università di Torino, 10095 Grugliasco (TO), Italy

**) Istituto Virologia Vegetale-CNR, Sezione Viticoltura, 10095 Grugliasco (TO), Italy

grapevine, two-dimensional polyacrylamide gel electrophoresis (2-DE)

The study of proteome is an innovative tool to help answer questions of functional analysis. The proteome, unlike the genome, is not a fixed feature of an organism. It changes with the state of development, the tissue, and even the environmental conditions under which an organism finds itself.

The study of proteome is possible by two-dimensional electrophoresis (2-DE), this method is capable of simultaneously separating hundreds of proteins. These several hundreds of proteins spots can be considered like genetic and physiological markers.

The availability of standardized and reproducible procedures for the analysis of proteins by 2-D electrophoreses is needed for this kind of investigation. The purpose of this research is to establish a routine procedure for the application of proteomic tools to the grapevine.

Experiments were made to set up reliable methods for proteomic analysis of grapevine tissues. The grape material used for analysis consisted of callus cultures grown on agar medium, of young leaves and of berries collected from field plants in the pre-veraison stage. The grape tissues used belonged to the ‘Nebbiolo’ variety.

Different techniques of protein extraction were tested through the use of extraction buffers containing Tris, urea, thiourea and different reducing agents.

The method which yielded the best results was :

protein extraction in lysis buffer containing 7M urea, 2M thiourea, 1%DTT, 2% PVPP, 2% Triton.X-100 and 50 mM Tris-HCl pH 7,5;

precipitation of the sample by TCA-acetone.

Using this method, we obtained electropherograms presenting well-defined spots within the window delimited by isoelectric points 3.5 and 8.5, and molecular masses of 15 and 180 kD.

The proteomic maps are in the process of being characterized by image analysis in order to detect differences in presence and quantity of the different proteins.