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ALTERNATIVE SPLICING AND BIOCHEMICAL FUNCTIONS OF COCOA CYSTATINS Pirovani, CP1,2, Santiago, A.S 1, Alvim, FC, Santos, LS, Gesteira, AS1, Margis, R2 ; Pereira, GAG3, Cascardo, JCM1 1 DCB/UESC, Bahia, Brasil;2CBIOT/UFRGS, 3 IB/UNICAMP, São Paulo, Brasil. Phytocystatins are cysteine-proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and defense mechanisms against pathogens. We identified four cystatins ORFs named TcCYS1, TcCYS2, TcCYS3 and TcCYS4. The cDNA encode 209, 127, 124 and 205 amino acid residues. Analyses of genes structures suggest that the TcCYS3 is an alternative splicing form of the TcCYS1 and represent family 2 cystatins . The four ORFs were sub-cloned, and His-Tag fused proteins expressed in E.coli. Polyclonal antibodies against the recombinant TcCYS1 and TcCYS4 were raised, allowing the immunodetection of the endogenous proteins in the young plant tissues. Recombinant proteins showed papain inhibition in gelatin/SDS-PAGE and colorimetric method with BApNA substrate. We examined the biochemical and structural properties of TcCYS4 under heat-stress conditions. The enzyme inhibitory reactivity was reduced by heating in a temperature-dependent manner and reached a minimal level by heating at 65 °C for 10 min. Size -exclusion chromatography showed that TcCYS4 forms a homodimer at 65 oC and that the reduction of inhibitory reactivity is due to the conversion of the monomeric to a dimeric form. The recombinant proteins revealed a toxic effect on the mycelium growth of Moniliophthora perniciosa. Similar toxic effect was observed against tobacco suspension cultures cells. The TcCYS3 and TcCYS4-sepharose-CNBr immobilized recruited different proteases isoforms in cacao and M. perniciosa extracts. Keywords: Theobroma cacao, plant-pathogen interaction, cisteine-protease Inhibitor, Moniliophthora perniciosa .