Validating housekeeping genes for quantitative plant gene expression studies in sunflower senescence process

FERNANDEZ, P.; DOSIO, G.A.A.; DI RIENZO J.; AGUIRREZÁBAL, L.A.; HOPP, H.E.; PANIEGO N.; HEINZ, R.A.

Salvador BA, Brasil

Congreso; 54° Congresso Brasileiro de Genética; 2008

Sociedad Brasilera de Genética

Leaf senescence is a regulated process that corresponds to the last stage in leaf development. It is characterized by dramatic changes in cellular metabolism and the sequential degeneration of cellular structures (Matile, 2000). Gene expression associated to these degradations may be considered as early changes in senescence process. Real-time PCR has been the largely and most fixed technique to quantify such instances. Besides being extremely powerful technique, real-time PCR suffers from certain pitfalls, most important being the normalization with a reference or housekeeping gene (Bustin and Nollan, 2004). It has been shown that abiotic stresses can induce or delay senescence (Buchanan-Wollaston et al. 2003) (Gan, 2003). The aim of this work was the validation of genuine housekeeping genes in sunflower leaves for real time PCR analysis under senescence-induced (water stress) or senescence-depressed (ablation of reproductive structure) treatments for this specie, and also for different aged leaves (from a medium and a high position in the canopy), given an important variability in the occurrence of early events related to senescence. A total of eight putative housekeeping genes were tested.. Two-step real-time RT-PCR was performed. The cDNA was synthesized from 1 ug of RNA using the Superscript first-strand synthesis system for RT-PCR (Invitrogen) with random hexamer primers according to manufacturer’s instructions. The real-time PCRs were performed using ABI Prism 7000 Sequence Detection System and software (PE Applied Biosystems, USA) with SYBR detection (QuantiTect SYBR Green PCR master mix (Qiagen, German). All the PCRs were performed under standardized conditions. The specificity of amplicons was verified by melting curve analysis (60 to 95° C) after 40 cycles and agarose gel electrophoresis. Three biological replicates for each sample were used for real-time PCR analysis and two technical replicates were analyzed for each biological replicate. The real-time PCR efficiency was determined for each gene and each stress with the slope of a linear regression model (Pflaff, 2001). To evaluate the gene expression stability, a mixed linear model was adjusted to the Ct values of every gene considered in this assay. The model included a stressing factor (3 levels) a leaf position within the plant factor (two positions) and the interaction between the stressing and leaf position factors. The model also included a random plant effect. Several statistics were calculated from the fitted model. Considering the fixed effects p-value it is clear that 26S rRNA, UBQ13, TUB2 and 18SrRNA, presents systematic variations between the stressing and/or position factors making them not appropriate as reference controls. Within the remaining genes, TUB1 seems to be the best according to plant’s variance component and CV value. In case it was decided to use more than one gene as a reference control TUB2, PEP and EF-1 could be the best selection.