Journal of Biotechnology 135 (2008) 105–116 Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Stress regulated expression of the GUS-marker gene (uidA) under the control of plant calmodulin and viral 35S promoters in a model fruit tree rootstock: Prunus incisa × serrula Fatemeh Maghuly a , Mahmood Ali Khan a , Eduviges Borroto Fernandez a , Philippe Druart b , Bernard Watillon b , Margit Laimer a,∗ a Plant Biotechnology Unit, Biotechnology Department, BOKU University, Muthgasse 18, 1190 Vienna, Austria b CRA, Biotechnology Department, Chaussee de Charleroi 234, 5030 Gembloux, Belgium article info Article history: Received 28 December 2007 Received in revised form 11 February 2008 Accepted 18 February 2008 Keywords: Salicylic acid Low-temperature High-temperature Wounding Real-time qPCR Gene expression abstract The fact that calmodulin genes (CaM) are tightly associated with the Ca 2+ regulatory pathway, as well as their putative role in plant defence against pathogens, indicate a potential use of alternative plant promoters to express genes of interest in specific tissues or developmental stages. To study the expression level of the apple CaM promoter, 981bp sequences upstream were fused to the uidA gene, introduced into cherry and compared with a 35S-GUS construct. Transgene copy number and transgenic expression levels were analysed using Southern blot, Western blot and RT-PCR techniques. Transcription levels were assessed by GUS fluorometry, histochemistry and real-time PCR techniques in leaves of plantlets grown in vitro under various abiotic stresses like low- and high-temperature, salicylic acid and wounding, harvested after 0, 0.5, 1, 2, 4, 10, 24 and 72h. Histochemical analyses showed staining only in veins and petioles of CaM-GUS lines, while in 35S-GUS plants staining extended to the entire leaf. Furthermore, real-time qPCR data indicate that both promoters are differently regulated by various stresses. Obtained results suggest that the selected apple CaM promoter responsible for the expression of a gene in vascular tissues may offer interesting perspectives for plant defense programs. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Regulation of transgenic expression is essential for the target gene at the desired level in specific plant tissues or organs. Different molecular factors control the expression of genes in plants, partic- ularly at the level of transcription, and the leading role belongs to a DNA sequence zone known as promoter (Potenza et al., 2004; Chen et al., 2007; Naumkina et al., 2007). Therefore, the investigation, isolation and characterization of promoters are highly desirable for controlling the expression of foreign genes in a particular tissue or the whole transgenic plant. The most widely used promoter for directing strong constitutive expression of the target gene in transgenic dicotyledonous plants is the Cauliflower Mosaic Virus 35S promoter (Roberts et al., 1992; Potenza et al., 2004; Xiao et al., 2005), which is generally active at high levels even in the absence of stress. In contrast, most promoters ∗ Corresponding author at: Plant Biotechnology Unit, Institute of Applied Micro- biology BOKU, Nussdorfer L¨ ande 11, A-1190 Vienna, Austria. Tel.: +43 1 36006 6560; fax: +43 1 36 97 615. E-mail address: m.laimer@iam.boku.ac.at (M. Laimer). of plant defensive genes are activated only after exposure to biotic or abiotic stresses. The application of native plant promoters can also help to avoid transgene silencing, which is often associated with the presence of promoters of non-plant origin in the plant genome (Matzke and Matzke, 1995; Yevtushenkoa et al., 2004). Although a wide range of plant constitutive promoters has been iso- lated and used for the generation of transgenic plants, there is still a strong demand for high-level expressing plant-derived promoters of transgenes (Potenza et al., 2004). Plants are surrounded by a variety of environmental stimuli that frequently impose constraints on growth and development. These stimuli include environmental signals produced by drought, salin- ity, cold, mechanical perturbation, hormonal signals, symbiotic and pathogenic microorganisms (Kiegle et al., 2000; Phean et al., 2005), which are the major limitations for plant productivity in cultivated areas worldwide. To overcome these limitations it is important to identify novel genes and their upstream regulatory regions. Also, the determination of gene expression patterns in response to stress and a better understanding of their functions in stress adapta- tion will provide the basis for an effective engineering strategy to improve stress tolerance in plants (Cushman and Bohnert, 2000; Hu et al., 2006). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.02.021