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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

REVERSE GENETICS ANALYSIS OF THE PRESENCE OF MTI-2 ANALOGUES IN THE BRASSICACEAE GENEPOOL AND THEIR IN VITRO EXPRESSION

PIGNONE D.*, ERRIQUEZ R.*, GALLERANI R.**, SONNANTE G.*, CECI L.R.***

* CNR-Istituto del Germoplasma, Bari

** Dip Biochimica e Biologia Molecolare, Un iversità di Bari

*** CNR-Centro Studi Mitocoindri e Metabolismo Energetico, Bari

Brassicaceae, proteinase inhibitors, molecular genetics, genetic resources, plant protection

Inhibitors of the proteinases have often been associated to seed resistance to insect attacks; trypsin inhibitors are particularly effective in starving the pests and thus in controlling their number. MTI-2 is a trypsin inhibitor isolated from Sinapis alba. The inhibitor is a small protein of 63 amino acids with a Ki (dissociation equilibrium constant) toward trypsin of 0.01 nM. The gene (mti-2) is discontinuous with the mature protein entirely encoded in the second exon.

From the gene nucleotide sequence, primers were derived to amplify the region encoding the mature protein. These primers were utilised in PCR amplifications using as template DNAs from various species of Brassicaceae (Brassica oleracea, Diplotaxis tenuifolia, D. muralis, ecc). The amplified fragments were cloned and single clones analysed for nucleotide sequence. The alignment of the sequences to the mti-2 gene allowed to identify the overall amount ofsequence similarity among the sequencesfor each amplification product, and the distribution of the areas with high homology. The translation in silico of the sequences and the comparison with the MTI-2 amino acid sequence primary structure allowed to demonstrated that the active site of the gene tends to beis conserved over a wide range of species although the general sequence of the each gene may change noteworthy.

Two inhibitors were also expressed in Pichia pastoris. and tTheir activity and the Ki toward against trypsin assayed by was measured throughing the hydrolysis of the synthetic BApNA substrate BApNA (N-Benzoyl-arginine-p-nitroanilide), allowing the measurement of Ki.

These described results open a new perspective in the analysis of genepools and for the search of novel active useful genes to be used in plant protection.

Work partly under the framework of MURST - Cluster C03 "Geni d'interesse biomedico ed agroalimentare" project and funded by Research Projects Program (Consorzio Interuniversitario per le Biotecnologie).e….