Caracterização da família multigênica da proteína desacopladora de plantas (pUCP) e regulação da expressão gênica sob diferentes condições de estresses abióticos em Vigna unguiculata (L.) Walp.

Abstract

The plant uncoupling proteins (pUCP) are located in the inner mitochondrial membrane and facilitate the proton translocation across the intermembrane space into the mitochondrial matrix, deflecting the passage of H + by F1-ATPase activity, thus affecting the efficiency of oxidative phosphorylation, i.e decreasing the synthesis of ATP coupled to the operation of the electron transport chain. Therefore, these proteins are responsible for dissipating the electrochemical gradient of H+, generated by respiration, releasing heat to the environment. These proteins belong to family of carriers Mitochondrial Anion (FCAM) and are encoded by multigene families. Their function is not yet fully elucidated, but the literature suggests involvement in adapting to biotic and abiotic stresses and cell protection by avoiding the production of reactive oxygen species (ROS). Their participation in adaptive thermogenesis is unclear. The aim of this work was to characterize the multigene family of pUCP in Vigna unguiculata (L.) Walp and your regulation through gene expression in response to different abiotic stresses. Vigna unguiculata seeds were germinated on paper imbibed water in the dark. Three days after germination the seedlings were transferred to Hoagland solution for 3 days before application of stress. Roots and leaves were collected at 0, 6, 12 and 24 hours after addition of the respective 100 mM NaCl, 200.67 g / L PEG, 10 mM H2O2 and 5 mM salicylic acid to characterize the profile of multigene family expression of genes for pUCP by semiquantitative RT-PCR and real-time PCR (qPCR). Specific primers were designed based on the sequences of cDNA / gene identified in pUCPs Vigna unguiculata using the PerlPrimer tool. Amplification of cDNA of actin was used to normalize the data for RT-PCR semi-quantitative and three constitutive genes were used to normalize the data of qPCR: 2 to actin gene (actin 4 and 5) and a gene for factor alogament 1-α (EF1α). In silico analysis revealed that in Fabales order pUCP is encoded by at least six pUCPs genes presenting a duplication of the gene type 1 (1a, 1b) and a pUCP6 gene deletion. The multigene family of pUCP, consisting of six genes was then identified in Vigna unguiculata (VuUCP1a, VuUCP1b, VuUCP2, VuUCP3, VuUCP4 and VuUCP5). The alignment of amino acid and nucleotide sequences of the species of Fabales order including Vigna unguiculata, revealed three xv. conserved sequences called signal Energy Transfer Protein (SPTE), and four domains (or regions or sites) existence of specific true pUCPs in Vigna unguiculata. VuUCP1a gene was constitutively expressed in leaves and roots, contrasting with VuUCP1b, whose expression was modulated in a stress and tissue-dependent manner. The VuUCP1b expression increased in response to all treatments (PEG, NaCl, H2O2 and salicylic acid) in roots, whereas the expression in leaves did not increase in response to NaCl. VuUCP2 expression was inhibited in response to PEG in leaves. VuUCP4 showed constitutive expression in response to stresses in both tissues, while VuUCP3 and VuUCP5 showed induction of expression by various stresses depending on the tissue type. The identification of the multigene family of pUCPs in Vigna unguiculata and its gene expression profile in different tissues and stress conditions highlights a possible role of this protein in the adjustment of plants to environmental stresses.