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Carboxypeptidases and trehalases of insects

Grant number: 12/07140-0
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2012
Effective date (End): July 31, 2012
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Clelia Ferreira Terra
Grantee:Katia Graziella de Oliveira Rebola
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:11/51685-8 - Insect digestion: a molecular, cellular, physiological and evolutionary approach, AP.TEM

Abstract

Digestion in insects: a molecular approach, cellular, physiological and evolutionary. Upon realization that the digestive tract is a relatively large interface little protection between the insect and the environment, an understanding of intestinal function was considered essential to the development of new methods of control acting through the gut, such as the use of transgenic plants to control phytophagous insects. (A. Rev. Biochem. 61:87-111, 1992). Our laboratory chose to study in detail the strategy for insect model arranged in the relevant points of the phylogenetic tree, to produce generalizations (A. Rev. Ent. 35: 181-200, 1990, Comp. Biochem. Physiol. B 109: 1 -62, 1994; Comprehensive Insect Molecular Science, vol. 4, Oxford, Elsevier, p. 171-224, 2005). The results showed that the digestive process in insects is organized phylogenetically certain standards and allowed to raise hypotheses about the role of the peritrophic membrane (Arch. Insect Biochem. Physiol 47: 47-61, 2001) and membranes perimicrovillar of Hemiptera ( bugs, cicadas and aphids) (An. Acad. Bras. Cienc. 78: 255-269, 2006). The development of the sector is the expansion of knowledge of the molecular events underlying the physiological phenomena bowel. This project addresses all aspects within the subdivision: (a) Study of digestive enzymes, (b) Molecular Physiology intestinal (c) Molecular basis of different mechanisms of intestinal secretion of enzymes and (d) Evolution of digestive systems of insects . Digestive enzymes will be studied using procedures described in our review (already mentioned and more Insect Biochem. Molec. Biol. 35: 883-901, 2005, Acta Crys. F62: 750-752, 2006). The objective is to seek the causes of structural and kinetic peculiarities of the digestive enzymes that enable the insects to circumvent the defenses of plants. The following enzymes have their encoding cDNAs cloned, expressed and be subjected to recombinant proteins functional studies (site-directed mutation) and cristolográficos studies: trypsin and chymotrypsin P. American T. molitor, M. domestica and D. saccharalis, cathepsin L (CAL), and aminopeptidase N trehalase T. molitor, trehalase, b-glucosidase and laminarinase S. frugiperda, laminarinase flavolineata of th. The enzymes are then sequenced and characterized kinetically: CALs D. peruvianus, a-mannosidase T. molitor amylase and D. saccharalis. Models of molecular physiology of intestinal D. peruvianus, T. molitor, M. domestica and S. frugiperda will be formulated in two steps: (a) random sequencing of cDNA libraries intestinal generation of antibodies for the most interesting clones and their use in imunocitolocalizações to generate provisional hypotheses. (B) functional testing of hypotheses by suppressing the expression of proteins selected by RNAi and evaluation of the effects. The mechanisms of secretion of some enzymes in insects appear to include aspects that are not found in other animals (review). For these studies, scans of cDNA libraries intestinal expression with antibodies against cytoskeletal proteins microvilar (S. frugiperda), the apex cell (T. molitor) and microvillar (both insects) identify positive clones. These hypotheses bear secretion mechanism in two steps procedure, such as those mentioned for the study of the physiology molecular intestine. The development of the evolutionary hypothesis of the digestive systems of insects should include studies of digestive physiology of insects is not considered so far as cricket and walking stick and advance the study of membranes in perimicrovillar Thysanoptera.