FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / As proteÃnas desacopladoras de planta (pUCPs) sÃo proteÃnas integrais de membrana, localizadas na membrana mitocondrial interna, pertencentes a FamÃlia de Carreadores de Ãnions Mitocondriais (FCAM), responsÃveis pela dissipaÃÃo do gradiente eletroquÃmico de prÃtons, gerado pela respiraÃÃo, como calor. Na presenÃa de Ãcidos graxos livres (AGL), as pUCPs facilitam a reentrada de prÃtons do espaÃo intermembranar para a matriz, desviando-os da ATP Sintase, inibindo assim a fosforilaÃÃo oxidativa. A funÃÃo dessas enzimas ainda nÃo està completamente elucidada, mas a literatura sugere a sua participaÃÃo em processos de amadurecimento de frutos, adaptaÃÃo a situaÃÃes de estresses biÃticos e abiÃticos e proteÃÃo da cÃlula evitando a produÃÃo de espÃcies reativas de oxigÃnio (EROS). Sua participaÃÃo na termogÃnese adaptativa à questionÃvel. O objetivo do presente trabalho foi caracterizar a atividade enzimÃtica da pUCP de mitocÃndrias de hipocÃtilos de Vigna unguiculata (L.) Walp atravÃs de ensaios polarogrÃficos, identificar o(s) gene(s) das pUCPs, estudando a sua expressÃo em diferentes situaÃÃes de estresses abiÃticos. A atividade da pUCP foi avaliada em presenÃa de Ãcidos graxos (palmÃtico, linolÃico, mirÃstico e lÃurico) e BSA tendo succinato e malato como substratos e distintos pHs (6,5, 7,2 e 7,8). As maiores atividades foram obtidas com Ãcido linoleico na presenÃa de succinato em pHs mais alcalinos (7,2 e 7,8). Primers degenerados a partir de dez diferentes pUCPs, denominados pump1 e pump2, foram desenhados para a amplificaÃÃo dos fragmentos gÃnicos por RT-PCR e PCR. Isolou-se o RNA total de folhas de plÃntulas de V. unguiculata submetidas a diferentes condiÃÃes de estresses (NaCl 100mM, H2O2 1mM e PEG 200,67g/L) que foram amplificados por RT-PCR, purificados e clonados no plasmÃdio pCR4-TOPO. Sete fragmentos gÃnicos de aproximadamente 760 pb foram seqÃenciados, apresentando 100% de identidade entre si. A comparaÃÃo da seqÃÃncia deduzida de aminoÃcidos desse fragmento de cDNA com a soja revelou 94% de homologia com GmUCP1a e 90% de homologia com GmUCP1b. Os resultados sugerem que o gene do feijÃo identificado em V. unguiculata (VuUCP1a) à ortÃlogo ao gene GmUCP1a de soja (Glycine max). A expressÃo de VuUCP1a em feijÃo em condiÃÃes de estresses abiÃticos (salino, oxidativo e osmÃtico) atravÃs de anÃlise por
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RT-PCR revelou um perfil diferencial, sugerindo induÃÃo de expressÃo de VuUCP1a apenas em resposta ao estresse salino. Isolou-se o DNA genÃmico de V. unguiculata e dois fragmentos gÃnicos (1700 e 1900pb) foram amplificados por PCR. A amplificaÃÃo de dois fragmentos distintos a partir do DNA genÃmico sugere a existÃncia de pelo menos dois genes codificando pUCPs em feijÃo, sendo assim, a pUCP Ã codificada por uma famÃlia multigÃnica. / The uncoupling proteins of plants (pUCPs) are integral proteins of membrane, located in the mitochondrial inner membrane, belong the Mitochondrial Anion Carrier Family (MACF). They are responsible for the dissipation as heat of the electrochemical gradient of protons, generated during respiration. In the presence of free fatty acid acids (FFA), the UCPs facilitate the re-entry of protons from intermembrane space for the matrix and these protons are deviated from the influence of the ATP sintase what leads to the inhibition the oxidative phosphorilation. The function of these enzymes completely is still not elucidated, but literature suggests its participation in processes of fruit maturation, in the adaptation to stress conditions and in cell protection by avoiding the production of reactive species of oxygen (EROS). The involvement of this enzyme in adaptive thermogenesis is questionable. The objective of the present work was to characterize the enzymatic activity of pUCP of mitochondria from hypocotyls of Vigna unguiculata (L.) Walp through polarographic assays and to identify the(s) gene(s) of pUCPs, through its expression in different conditions of abiotic stress. The activity of pUCP was evaluated in the presence of fatty acids (palmitic, linoleic, myristic and lauric) and BSA, having succinate and malate as oxidizable substrates at different pHs (6,5, 7,2 and 7,8). The higuest activities were obtained in the presence of linoleic acid, succinate as substrate and with more alkaline pHs (7,2 and 7,8). Degenerate primers, obtained from ten different pUCPs, called pump1 and pump2, has been designed for amplification of the gene fragments through RT-PCR and PCR. The total RNA was isolated from plants leaves of V. unguiculata submitted to different stress conditions (100 mM NaCl, 1 mM H2O2 and 200,67 g/L PEG) and cDNAs fragments amplified by RT-PCR had been purified and cloned in the plasmid PCR4-TOPO. Seven cDNA fragments of approximately 760 pb had been sequenced and presented 100% of identity among themselves. The comparison of the deduced amino acid sequence of this cDNA fragment with those of soybean disclosed 94% of identity with the gene GmUCP1a and 90% of identity GmUCP1b soybean gene. These results suggest that the pUCP gene identified in V. unguiculata (VuUCP1a) is ortologous to the GmUCP1a gene from soybean (Glycine max). The expression of genes of pUCP in beans, under abiotic stress
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conditions (salinity, oxidative and osmotic conditions) analyzed through RT-PCR disclosed a differential profile what suggests induction of expression of VuUCP1a only in response to salt stress. The genomic DNA of V. unguiculata was isolated and two gene fragments (1700 and 1900 pb) had been amplified by PCR. The amplification of two fragments from the genomic DNA suggests the existence of at least two pUCPs genes in beans what leads to the conclusion of a pUCP multigenic family.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:2010 |
| Date | 26 April 2007 |
| Creators | Francisco Edson Alves Garantizado |
| Contributors | Dirce Fernandes de Melo |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em BioquÃmica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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