Reference gene selection for Real-Time PCR in Bidens laevis L. during pesticide and cold stress

LUKASZEWICZ, GERMÁN; AMÉ, MARÍA VALERIA; MENONE, MIRTA

Congreso; 6th SETAC World Congress / SETAC Europe 22nd Annual Meeting; 2012

Environmental monitoring demands the usage of new species and sensitive methods in order to get accurate regional biomonitoring. The aquatic macrophyte Bidens laevis L. (fam.: Asteraceae) has desirable growing characteristics for use in laboratory assays, has proven to respond positively to genotoxic compounds and showed enzimatic response when exposed to the pesticide endosulfan. Nowadays, gene expression offers crucial information when analysing responses to xenobiotic compounds. It is well known that Real Time PCR (RT-qPCR) is the preferred method for studing gene expression because of its sensitivity, precision and robustness but it requires the use of reference genes with stable expression among different treatments, as well as different tissues and growth stages. The aim of this study was to analize the expression of two candidate genes, Elongation Factor 1 alfa (EF1a) and Actin (ACT) in B. laevis in three tissues (root, stem and leaves) under four conditions: 1-control-: Large plants (>1500mg), temperature 22¨¬C, media: Hoagland solution; 2 -short-: Short plants (<250mg), temperature 22¨¬C, media: Hoagland solution; 3 -xenobiotic-: Large plants, temperature 22¨¬C, media: Hoagland solution + endosulfan 10ug/L; 4 -cold- Large plants, temperature 5¨¬C, media: Hoagland solution. All plants remained 24hs under each condition with 3 replicates tested for each one. Firstly, the partial sequences for both genes were obtained from degenerate PCR products. The two sequences showed an identity of 82% (EF1a) and 81% (ACT) compared to other members of Asteraceae family. From these sequences a set of primers for each gene was designed and the RT-qPCR was optimized. The results showed that EF1a had no significant variation in expression among the three different tissues. This expression remained stable between control and short plants. However, a significant expression increase was observed in plants exposed to endosulfan or cold. In contrast, ACT expression did not show significant differences among tissues or under any condition tested in this study. From these results we can conclude that Actin expression did not seem to be influenced in short plants or during endosulfan or cold exposure and can thus be used as a reference gene for RT-qPCR in any tissue, while Elongation Factor-1 alfa should not be used when analysing stress conditions such as cold and xenobiotic exposure.