Isolamento e caracterização dos genes que codificam malato sintase e o repressor Nrg1 em Crinipellis perniciosa, agente causal da vassoura-de-bruxa no cacaueiro (Theobroma cacao)

• Autor(a) principal: Medina, Pilar Ximena Lizarazo
• Data de Publicação: 2006
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1565
• topic: Repressão catabólica; Malato sintase; Crinipellis perniciosa; Catabolic repressor; Malate sintase; Crinipellis perniciosa; CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS

The genes codifying for either the catabolic repressor Nrg1 and malate synthase enzyme from Crinipellis perniciosa were cloned and characterized. The codifying? coding? region of the nrg1 gene has 2121 pb and is interrupted by a 60 pb intron as indicated by cDNA sequence analysis. Total DNA blot analyses revealed the gene to be as a single copy in the genome of C. perniciosa. The deduced protein shows two characteristic zinc fingers that are characteristic for Nrg proteins, and has 256 amino acids. The terminal 5 region was sequenced, whereas a putative cis-element for binding the PacC repressor was identified. The cis-elements TATA box and CAAT box were also identified. The transcription of nrg1 was investigated, by applying the RT-PCR technique. The gene was differently regulated according to glucose concentration. The nrg1 transcription was 2.1-fold greater in the mycelia grown in a medium containing 0,1.% glucose than in medium with 3.0% glucose. In other carbon source such as glycerol, the gene was transcripted 4.6-fold greater than in 3.0% glucose. The evaluation of the gene transcription relative to pH pointed out a higher expression at low pH (2 and 3) than at pH values 4, 6.8, and 8. Those results are explained by the presence of the sites for the regulating protein PacC, which is responsible for regulation in response to pH. This is the first report about the presence of this repressor protein Nrg1 in filamentous fungi. The second gene under study codes for the malate synthase enzyme from C. perniciosa. The gene has 1888 pb and is interrupted by five introns. A protein with 539 amino acid residues was obtained from the translation of this gene and exhibits the characteristic sequence [KR]-[DENQ]-H-X(2)-G-L-N-X-WD-Y-[LIVM]-F. A possible TATA box and two possible CAAT sites were located on the 5 flanking region of the malate synthase gene. The DNA hybridization of isolates from C. perniciosa of the groups C1, C2, C3 and C9 with the malate synthase gene showed this one to be present in a single in genome. It is interesting that this hybridization allowed for separating the isolates into two groups. In isolates C1 and C3 collected in Bahia and Amazonas states, the hybridized fragment has 5.0 kb approximately, but the isolates C2 and C9 proceeding from Bahia and Pará states this fragment has 4.5 kb. The transcription of the malate synthase gene was analyzed, by using the technique RT-PCR. This transcription was analyzed and the mycelia grown at 27 ºC for 8 days were used. Then, the mycelia were washed and transferred to a medium containing acetate, isocitrate, glyoxylate in both presence and absence of glucose, where they were kept for 12 or 24 hours. There was observed the absence of the catabolic repression. Also, the transcription of the malate synthase gene was observed in glycerol and in the cocoa pulp extract. The isolation and characterization of the genes codifying the malate synthase and the regulating protein Nrg1 in C. perniciosa together with the availability of the transformation system made possible the obtainment of mutants to evidence the paper of the proteins in pathogenicity.