Selection and validation of reference genes for functional studies in the Calliphoridae family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann. The expression stability of six genes (Actin, Gapdh, Rp49, Rps17, alpha-tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.14Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2008/58106-0]CNPq [477335/2009-8]FAPESP [2009/13463-3

Functional Dissection of Sugar Signals Affecting Gene Expression in Arabidopsis thaliana

Background: Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. Methodology/Principal Findings: To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants. Conclusions: Altogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism