Caracterização das interações moleculares entre lectinas manose específicas tóxicas e o trato digestivo do pulgão da ervilha, Acyrthosiphon pisum (Harris)

Abstract

Lectins isolated from Diocleinae (Fabaceae) subtribe seeds present very similar structural and physicochemical properties. In this Thesis, we used the mannose/glucose-specific Canavalia brasiliensis (Con Br) lectin as a tool to isolate and identify molecular targets for lectins in the digestive tract of the pea aphid Acyrthosiphon písum Harris (Homoptera: Aphididae). In vitro toxicity assays were performed with-16 lectins including 9 from the Diocleinae subtribe. Statistical analysis of the mortality and growth inhibition curves show that all the Diocleinae lectins, with the exception of Dioclea grandiflora, exert toxic effects on the development of the insect. Con Br and lectins from Canavalia bonariensis, C.aff.sp.boliviana, Dioclea virgata and D.rostrata were found to be the most toxic ones, decreasing the average weight of the adult insects by 50 % in concentrations ranging from 100 to 122 μg.mL- '. The lectin from Abrus pulchellus was found to be extremely toxic, being able to kill 100% of the insects at their fourth day of development at a low dose (500 μg.mL-1)_ Lectin blot assays were performed on aphid gut protein extracts using biotin-labeled lectins from the Diocleinae group, such as Con Br, and lectins from C.ensiformis, Cratylia floribunda and D. violacea, and from monocotyledon group, such as from Galanthus nivalis, Listera ovata and Arum maculatum. Several proteins from the digestive tract of A.pisum were strongly recognized by both lectin groups. Moreover, quantitative differences were observed between groups and also within the monocotyledons group. Several proteins from the digestive tract of the insect were isolated by means of gel affinity chromatography, with Con Br-Agarose, followed by non-native two-dimensional polyacrylamide gel electrophoresis. Further studies performed on the isolated proteins allowed us to identify an aminopeptidase of apparent molecular weight 120 kDa and pl 5.0. Its partial nucleotide sequence was determined using a genomic library constructed from messenger RNA extracted from A.pisum digestive tract. The role of the aminopeptidase in the toxic effects exerted by the abovementioned lectins is discussed in this Thesis.