Avaliação da região promotora do gene da trealase ácida atm1 e metarhizium anisopliae para construção de vetor regulável por fonte de carbono

Abstract

Fungal expression systems have been used in several functional analysis approaches as tools for altering gene expression. In Metarhizium anisopliae, however, few studies have been conducted regarding efficient homologous promoter and their use in constructing expression systems that may facilitate such approaches. This biocontrol entomopathogenic fungus is widely known for its ability to infect a wide range of economically important arthropod, from farming pests to disease vectors. During the infection process, M. anisopliae directly penetrates the host cuticle using multifactorial resources that include the mechanical pressure exerted by appressorium and the secretion of hydrolases, among others. In one of the stages of infection, when reaching the hemolymph of the host, this fungus differs into blastospores and is disseminated in the host by circulatory system. The hemolymph of arthropods has as main component, the disaccharide trehalose. To intake and use this nutrient, the fungus secretes acid trehalase, which hydrolyses trehalose into two glucose units. In a genomic analysis, a putative ortholog of the gene encoding this hydrolase (ATM1) was identified in M. anisopliae. However, there are few reports in literature characterizing the 5' UTR region of this gene, as well as regulatory elements that may give indications of its regulation. It is suggested that elements involved in catabolic repression by glucose are present, such as trehalose responsive elements. Understanding the mechanisms involved in promoter regulation is important not only for the basic knowledge of its functioning, but also for development tools for studying gene function. Regulable expression vectors are molecular tools useful in the investigation of gene function and their differential is to allow the regulation of the promoter in order to obtain the most precise control of the gene expression under study. The promoter of the acid trehalase, in this study probably, is only induced in host hemolymph, which allows the control of the expression of interest genes in media that mimic hemolymph conditions, especially the presence of trehalose. This work analyzed the promoter region of the acid trehalase gene of M. anisopliae and evaluated its regulation by carbon source. Strains containing cassettes inserted into the E6 strain of M. anisopliae genome containing the reporter genes green fluorescent protein (GFP) and red fluorescent (yeCherry) were obtained under the regulation of different sequences of the putative promoter region of interest. Transformants were analyzed for induction of reporter gene expression with trehalose and repression with glucose quantitatively by mRNA analysis (RT-qPCR). Was possible to identify a minimal functional promoter region of 750 bp that controls the expression of reporter genes and may be induced by trehalose. These results demonstrate that the M. anisopliae homolog acid trehalase promoter is regulated by the carbon sources tested and offer a new tool for gene function studies.