Pré-validação de promotor tecido-específico de raiz e genes de resistência a fusariose em Nicotiana tabacum

Abstract

The production of genetically modified plants with specific characteristics is a highly promising process for plant genetic breeding that have been severely threatened by diseases such as fusarium wilt, for example, which causes great economic losses. In addition to the data mining of numerous candidate genes that could be used for teste disease resistance, the lack of tissue-specific promoters that allow these genes to be expressed only in target tissues, increase the population's distrust of plant genetically modified. Research on the use of tissue-specific promoters is important, since they drive the genes expression only in the organ/tissue of interest, avoiding expression in all parts of the plant, which in addition to having a high energy cost, can cause undesirable phenotypic effects. The main of this work was to evaluate in vitro the effectiveness of a root tissue-specific promoter and the functionality of two resistance candidate genes (Foc1 and Foc2) against fusarium wilt through genetic transformation of a model plant (Nicotiana tabacum) using Agrobacterium tumefaciens. In the results of the expression analysis of GUS reporter gene under the regulation of the tissue-specific promoter (Prom_Musa_Embrapa_005) in a core version (pCore) it was possible to confirm their specificity to the root, once the histochemical assay to detect the enzymatic activity of β-glucuronidase (GUS) made in leaf and root tissues of transformed N. tabacum, showed activity only in root, especially in the root cap, demonstrating the potential to provide a focused expression. In the gene expression analysis by qPCR it was also possible to verify the specific expression of the GUS gene in the roots of transformed plants with the promoter and the constitutive expression of the positive control as well as with the candidate resistance gene (Foc1). The constitutive expression of Foc1 occurred as expected because the gene was under CaMV-35S promoter regulation. From this analysis it is possible to confirm that the plants were effectively transformed with the resistance gene. Foc1 presents potential to be introduced into cultivars of interest, making possible the development of fusarium wilt resistant varieties. This study will support the preparation of new constructs with tissue-specific promoters regulating Foc1 gene expression.