Quitinase de Classe I de feijão-de-corda (Vigna unguiculata): Estudo preliminar da expressão do gene, clonagem, expressão e purificação em Escherichia coli BL21(λ)DE3 e determinação da estrutura através da modelagem por homologia

Abstract

In this work we have made a preliminary study on the expression of a class I chitinase gene from cowpea (Vigna unguiculata L.Walp), cloning and expression of this gene in Escherichia coli BL21(λ)DE3 cells and, through homology modeling, we determined the three­ dimensional structure of this protein. The expression of the class I quitinase gene from cowpea was performed by RT­PCR from the total RNA, with specific primers, from seeds and pods in distinct stages of development (2, 4, 6, 8 , 10, 12, 14, 16 and 18 days), belonging to two contrasting genotypes regarding to infection by Callosobruchus maculatus, IT81D­1053 (resistant) e TE97­419­07F (susceptible). The gene expression in leaves, roots, epicotyls and hipocotyls from two contrasting genotypes considering the infection by the nematoid Meloidogyne incongnita, CE­31(resistant) e TE97­411­1F (susceptible). The VuChiI gene cloning was accomplished from the amplified product on RT­PCR with IT81D­1053 seeds, and the amplified product from the genomic DNA of MONTEIRO. Eleven clones were obtained, from which nine were sequenced. The cloning of R7 clone was accomplished in pET15b vector and the expression of the recombinant protein was induced in the presence of IPTG 1mM. The protein, with 30 kDa, was visualized through a SDS­PAGE. The protein purified through an affinity chromatography in Sepharose column with immobilized Nickel did not had a significative hydrolytic activity. The models generated for the clones and for the native chitinase, have indicated that mutations that occurred did not changed the molecule's active sites. Thus, the class I chitinase gene from feijão­ de­ corda seems to present constitutive expression in all parts of the plant. The recombinant chitinase obtained was inactive. The specific mutations in the resulting clones suggests the occurrence of isoforms of this protein, what should be elucidated in the future.