Efeito do estresse hídrico/salino na expressão da oxidase alternativa de Vigna unguiculata (L.) Walp e produção da proteína recombinante

Abstract

Higher plants, fungi, some algae and protists possess an alternative electron transport pathway, not coupled to ATP synthesis, KCN-insensitive, mediated by alternative oxidase (AOX). In some plants, the alternative oxidase is encoded by a multigene family and its expression is induced by several stress types often related to the production of free radicals. The present work was carried out in three different aspects: 1. Expression of AOX in cultivars of Vigna unguiculata L. walp with different resistance degrees to water/salt stress: cv. Vita 3 (more resistant) and cv. Vita 5 (less resistant); 2. Characterization of genes of AOX of V. unguiculata, starting from a cDNA library XZIPLOX of leaves of the cv. EPACE1; 3. Description of the production of a recombinant protein in Escherichia co/. Dark-grown V. unguiculata seedlings were transferred into a hydroponic system, under light conditions, after the third day of germination. After the tenth day, an osmotic shock was applied through the addition, in the nutritive solution, of Polyethylene glycol (PEG) or NaCI under the corresponding concentrations: PEG 200,67 g/L or 276,28 g/L and NaCI 100 mM or 200 mM. After seven days of stress roots and leaves from Vita-3 and Vita-5 cultivars were collected in order to purify mitochondria, to isolate total RNA and to prepare crude protein extract. The amount of AOX protein was evaluated through "Western blotting" carried out with the mitochondrial fraction and crude protein extract. The quantification of the transcripts was done through RT-PCR and "Northern blotting". A fragment of cDNA, homologous to conserved regions, was used as probe in the " screening" of the cDNA library and the recombinant protein was produced starting from the cDNA Vu-clone 5 in a vector of expression pQE-30. In roots, the expression of AOX was higher in Vita 3 than in Vita-5, in all the studied conditions, either with proteins or transcripts revealed by RT-PCR with "primers" of conserved regions. The band corresponding to the AOX reduced form (ca. 35 kDa) increased with the stresses in crude protein extract of Vita 3 roots and such increase was confirmed by the transcripts, revealed by hybridization with the probe Vu-clone 5. In root mitochondria of both cultivars, an intense band corresponding to the oxidized form of AOX (ca 70 kDa) was observed in all studied conditions and the band corresponding to the reduced form of AOX increased in the stress conditions. Vita 5 roots presented an increase in the amount of the AOX protein of 85 and 45% in the stresses, respectively induced by NaCI 100 mM and PEG 200,67 g/L. A similar tendency was not observed with the transcripts, revealed with the probe Vu-clone 5. Thirteen positive clones of the AOX were isolated from the cDNA library and two different genes were characterized, Vu-clone 3 and Vu-clone 5 in the proportion of 9:4. The recombinant AOX encoded by Vu-clone 5 was purified by Ni-NTA affinity chromatography and detected with monoclonal antibodies against AOX. The results show that the V. unguiculata AOX is encoded by a multigene family of, at least, two distinct genes whose expression is induced, in a different way, in each cultivar with different resistance degrees to water/salt. These data reinforce the suggestion that the AOX participates in the adjustment mechanisms of the plants submitted to unfavorable environmental conditions.