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Promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs developmental-regulated, stress-inducible, and organ-specific gene expression in transgenic tobacco

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Abstract

In our recent published work it has been demonstrated that AlSAP, a gene encoding an A20/AN1 zinc-finger protein (stress-associated protein) of the C4 halophyte grass Aeluropus littoralis, is inducible by various abiotic stresses and by hormonal stimuli. To further investigate the regulation of the gene, a 586-bp genomic fragment upstream of the AlSAP translated sequence has been isolated, cloned, and designated as the “Pr AlSAP ” promoter. Sequence analysis of “Pr AlSAP ” revealed the presence of cis-regulatory elements which could be required for abiotic stress, abscisic acid (ABA), and salicylic acid (SA) responsiveness and for tissue-specific and vascular expression. The Pr AlSAP promoter was fused to the β-glucuronidase (gusA) gene and the resulting construct transferred into tobacco. Histochemical assays of stably transformed tobacco plants showed that Pr AlSAP is active in this heterologous C3 system. While full-length gusA transcripts accumulated in whole 15, 30, and 45-day-old plants, GUS histochemical staining was only observed in leaves and stems of 45-day-old, or older, transgenic seedlings. Histological sections prepared at this stage revealed activity localized in leaf veins (phloem and bundle sheath) and stems (phloem and cortex) but not in roots. Furthermore, gusA transcripts accumulated in an age-dependent manner with a basipetal pattern in leaf and stem tissues throughout the plant. In flowers, GUS expression was detected in sepals only. The accumulation of gusA transcripts was up-regulated by salt, dehydration, ABA, and SA treatment. Altogether, these results show that, when used in a heterologous dicot system, Pr AlSAP is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter.

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Acknowledgments

The authors are grateful to S. Abid a teacher of English, for the English revision. Part of this work was conducted under the REFUGE platform funded by Agropolis Foundation, Montpellier France. This study was supported by a grant from the Ministry of Higher Education Scientific Research of Tunisia (contract program 2006–2010) and by the European project CEDROME (FP6-INCO-CT-2005-015468).

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  1. University of Sfax, Centre of Biotechnology of Sfax (CBS), BP“1117”, 3018, Sfax, Tunisia

    Rania Ben Saad, Walid Ben Romdhan, Nabil Zouari, Jalel Azaza & Afif Hassairi

  2. CIRAD, UMR DAP, Avenue Agropolis, 34398, Montpellier Cedex5, France

    Delphine Mieulet, Jean-Luc Verdeil & Emmanuel Guiderdoni

  3. King Saud University, CEBR, PO Box 2460, Riyadh, 11451, Saudi Arabia

    Afif Hassairi

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  1. Rania Ben Saad
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Correspondence to Afif Hassairi.

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R. Ben Saad and A. Hassairi are the first authors, and contributed equally to this work.

Accession numbers: AlSAP-DQ885218; Pr AlSAP -DQ885219.

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Online Resource 1

The third High-Efficiency Thermal Asymmetric Interlaced (HE-TAIL) PCR for 5′-flanking sequence of AlSAP gene. M: DNA Ladder; lanes 1, 2 and 3: products of the tertiary PCR using Rn2-ZFRv3, Rn2-ZFRv4 and Rn2-Rn2 primers, respectively (TIFF 68 kb)

Online Resource 2

Southern blot analysis of transgenic lines (L4, L6, L8 and L12). Genomic DNA was extracted, digested with NcoI (cuts once in the T-DNA) and loaded onto the gel which was blotted and probed with labeled PCR product of gusA gene (TIFF 56 kb)

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Saad, R.B., Romdhan, W.B., Zouari, N. et al. Promoter of the AlSAP gene from the halophyte grass Aeluropus littoralis directs developmental-regulated, stress-inducible, and organ-specific gene expression in transgenic tobacco. Transgenic Res 20, 1003–1018 (2011). https://doi.org/10.1007/s11248-010-9474-6

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