Artículos de revistas
Reference Genes For Quantitative Rt-pcr Of The Pilocarpine Producer Pilocarpus Microphyllus And Two Other Pilocarpus Species
Registro en:
Theoretical And Experimental Plant Physiology. Brazilian Soc Plant Physiology, v. 29, p. 51 - 59, 2017.
2197-0025
WOS:000398718000005
10.1007/s40626-016-0080-8
Autor
Franco
Mariana Crotti; Schimpl
Flavia Camila; Mazzafera
Paulo
Institución
Resumen
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Members of the genus Pilocarpus are the only natural source of the imidazole alkaloid pilocarpine, a plant metabolite widely used in the pharmaceutical industry. Although there is variation in the pilocarpine content in Pilocarpus, only the species Pilocarpus spicatus does not have pilocarpine, although it contains other imidazole alkaloids related with pilocarpine. Transcriptome and metabolome analyses have been conducted aiming to identify candidate genes related with the biosynthesis pathway of pilocarpine. Thus, the use of quantitative RT-PCR is essential for a rapid and effective analysis of the expression of candidate genes. Consequently, an accurate normalization of the experiments with stable reference genes is needed in order to minimize possible errors in the expression analysis. Eight reference genes (EF1a, UBQ, ICDH, GAPDH, ACT, TUBa, CYC, SAND) were assayed in leaf samples of three species of jaborandi (P. microphyllus, P. spicatus and P. pennatifolius), which were analyzed individually and together. The stability data were obtained with the softwares Normfinder, geNorm and BestKeeper. When the three species were analyzed together the most stable pair of genes was GAPDH and ACT. When analyzed individually, different genes were selected for each species. It was possible to determine the best reference genes for each one of the species of jaborandi evaluated and for the dataset of the three species. Additionally, with the use of geNorm, it was possible to establish the minimum number of reference genes necessary for a qPCR experiment. The gene coding for the small subunit of RuBisCo was used to validate the genes selected by the three softwares. 29 1 51 59 Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)