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

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 of sequence similarity among the sequences for 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 be is 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 through ing 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.