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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

A NEW GENE FAMILY CODING FOR SERINE PROTEINASE INHIBITORS IN RAPESEED

DE LEO F., VOLPICELLA M., GALLERANI R.

Dip. Biochimica e Biologia Molecolare, Università di Bari

One of the main problems linked with plant biotechnologies is related to the environmental impact of transgenes used for increasing plant productivity and resistances to plant biotic and abiotic stresses.

Among the strategies used for increasing plant resistances, that based on the transfer of enzyme inhibitor genes is, at the moment, the most "biological" available. The only relevant limitation of this strategy concerns with the insect adaptation, which can be overcome transferring contemporaneously more genes coding for inhibitors having complementary effects.

Two species of Cruciferae, mustard and rapeseed, contain new kind of serine proteinase inhibitors (PIs) highly homologous to each other, known as Mustard Trypsin Inhibitor-2 (MTI-2) and Rapeseed Trypsin Inhibitor (RTI-III). These inhibitors cannot be catalogued among the families of plant PIs described so far. Tentatively they can be considered the members of a new family of plant PIs.

This report focus on the identification of MTI-2 analogous in rapeseed. The presence of several serine proteinase inhibitors in rapeseed was confirmed by activity gels on total protein extracts. Only one of them cross reacts with antibodies against recombinant MTI-2. A probe containing part of the mti-2 gene has been used for identifying the homologous genes on the rapeseed genome. Three genes have been sequenced and one of them corresponds to the RTI-III protein already characterized by other authors. The transcripts of each gene have been detected in seeds imbibed for 24h. The genes consist of two exons with the mature protein coded entirely in the second. Two of the three deduced protein (RTI-I and RTI-III) have the reactive site typical of trypsin PI (Arg-Ile) whereas RTI-II has a Glu residue instead of Arg. The RTI-II has been expressed in P.pastoris yeast and studies on its activity against proteinases of different origins are in progress.

Acknowledgements-This work was funded by the Research Projects Program of C.I.B. (Consorzio Interuniversitario per le Biotecnologie).