These nuclear-encoded chloroplast
proteins are synthesised by cytoplasmatic ribosomes and transported post-translationally into the chloroplast. Some of them are assembled with the plastid-encoded PF-04929113 proteins to form functional complexes (e.g. Rubisco, ATP-synthase). For reliable measuring, the expression levels of photosynthetic genes, which can be nuclear- or plastid-encoded, selection of multiple appropriate reference genes for normalisation is very important. Gene expression levels have commonly been determined using northern blot analysis. However, this technique is time-consuming and requires a large quantity of RNA (Dean et al. 2002). The most widely used mRNA quantification methods nowadays are real-time fluorescence detection assays (Heid et al. 1996), due to their conceptual simplicity, sensitivity, practical ease and high-throughput capacity (Vandesompele et al. 2002; Bustin 2000). Mostly, normalisation of gene expression has been studied by using one selected MK-4827 “housekeeping gene” which is involved in basic cellular processes, and which is supposed to have a uniform level of expression across
different treatments, organs and developmental stages (Vandesompele et al. 2002). However, many studies have shown that the expression of these “housekeeping genes” can vary with the experimental conditions (Czechowski et al. 2005; Thellin et al. 1999; Gonçalves et al. 2005). Furthermore, as a new standard in real-time PCR, at least two or three housekeeping genes should be used as internal standards,
because the use of a single gene for normalisation can lead to large errors (Thellin et al. 1999; Vandesompele et al. 2002; Gutierrez et al. 2008). Studies on the identification of multiple reference genes mainly deal with human ever tissues, bacteria and viruses. Only a few publications exist for plants: for potato under biotic and abiotic stress (Nicot et al. 2005); for rice under hormone, salt and drought stress (Kim et al. 2003); for Arabidopsis selleck chemicals thaliana and tobacco under heat-stress and developmental changes (Volkov et al. 2003); for maritime pine during embryogenesis (Gonçalves et al. 2005) and for Arabidopsis thaliana under different environmental conditions and developmental stages (Czechowski et al. 2005; Remans et al. 2008). Reference genes for normalisation of plastid-encoded genes have not yet been determined. We selected from previous reports and micro-array data five nuclear-encoded and nine plastid-encoded reference genes and evaluated these in transgenic tobacco plants with increased (Pssu-ipt) and diminished cytokinin (35S:AtCKX1) content and their respective wild types, using the geNorm (Vandesompele et al. 2002) algorithm.