Identificação, análise in sílico de genes e proteínas e elaboração de estratégias de isolamento de genes potencialmente envolvidos na biossíntese e inativação de 20- Hidroxiecdisona (20e) em plantas.

Abstract

The production of secondary metabolites is one of the main defenses of plants against herbivorous insect attacks. The 20-hydroxyecdysone (20E) molecule is a hormone produced by insects, whose function is related to several processes during insect development. Besides the production in insects, some plants are also capable of synthesizing 20E, possibly using it as part of the defense mechanism during herbivory. The characterization and isolation of genes and proteins involved in plant defense mechanisms is essential to develop strategies aimed at controlling insects that attack species of economic interest, however the 20E biosynthesis route is partially known in insects and unknown in plants. The 20E molecule also has pharmacological, medicinal and biotechnological applicability. In this context, the objective of this work was to carry out the identification and in silico analysis of genes and proteins and to elaborate strategies for the isolation of genes potentially involved in the biosynthesis of 20E in plants. For this, a search for similar proteins in a database was performed using the BLAST/NCBI tool using as a basis the amino acid sequence of proteins that act in the partially known biosynthesis route (Neverland, Shade, Shadow, Disembodied and Phantom proteins) and inactivation (Ecdysone oxidase, 3-Dehydroecdysone 3beta-reductase and 3-Dehydroecdysone 3alpha-reductase proteins) of 20E in insects. 239 potentially homologous proteins were identified and analyzed in silico, with similarity values between 25 and 45%, high coverage values and an E-value of at least e-5. Pair and multiple alignments, prediction of physicochemical values (molecular weight, theoretical isoelectric point, number of negative and positive residues, extinction coefficient, instability index, aliphatic index and hydrophobicity index), subcellular location and phylogeny were performed. Shade, Shadow, and 3DE 3beta-reductase-like proteins were detected in higher amounts and in several plant species, while Disembodied and Neverland-like proteins were detected in one and two species, respectively. Strategies aimed at the isolation of genes potentially involved with 20E biosynthesis in plants have been proposed through the use of oligonucleotides, constructed from conserved regions, obtained through multiple nucleotide sequence alignments of genes. Several proteins found in vegetables have a function that is still unknown and may be involved in the biosynthesis of 20E in plants; however, its involvement and actual role still need to be analyzed.