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Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene

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Abstract

Plant calcium-dependent protein kinases (CDPKs) are involved in calcium-mediated signal transduction pathways. Their expression is finely tuned in different tissues and in response to specific signals, but the mechanism of such a regulation is still largely unknown. OsCDPK2 gene expression is modulated in vivo during rice (Oryza sativa L.) flower development and is downregulated by white light in leaves. In order to identify OsCDPK2 regulatory sequences, we amplified and cloned both the 5′ and 3′-flanking regions of the gene. Sequence analysis revealed that the leader sequence is interrupted by an intron, whose regulatory role was investigated. Different ß-gucuronidase (GUS) expression vectors, carrying combinations of the putative OsCDPK2 regulatory regions, were generated and GUS expression was analyzed both in transient assays and in transgenic rice plants. The whole 5′-flanking sequence was able to drive GUS expression in rice calli and leaves transiently transformed with the biolistic technique. Analysis of the GUS expression pattern in transgenic plants revealed strong activity in root tips, leaf veins and mesophyll cells, in flower reproductive organs and in mature pollen grains. Expression was also shown to be subject to an intron-mediated enhancement (IME) mechanism, since the deletion of the leader intron sequence from chimeric OsCDPK2::GUS plasmids almost completely abolished GUS activity. Furthermore, in transiently transformed leaves, GUS expression driven by the OsCDPK2 promoter-leader region was constitutively observed regardless of light or dark exposure. Light-regulated expression was restored by inserting the OsCDPK2 3′ untranslated region (3′UTR) downstream of the chimeric OsCDPK2::GUS transcription unit, suggesting that light down-regulation is mediated by a mechanism driven by the 3′UTR.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

CARE:

cis-Acting regulatory elements

CNS:

Conserved noncoding sequence

GFP:

Green-fluorescent protein

GUS:

ß-gucuronidase

IME:

Intron-mediated enhancement

NAA:

1-Naphtaleneacetic acid

NR:

Nitrate reductase

SPS:

Sucrose-phosphate synthase

UTR:

Untranslated region

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Authors and Affiliations

  1. Istituto Biologia e Biotecnologia Agraria, CNR, Via Bassini 15, 20133, Milano, Italy

    Laura Morello & Diego Breviario

  2. Dipartimento di Biologia, Sezione di Botanica Generale, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy

    Mauro Bardini, Mauro Cricrì & Francesco Sala

Authors
  1. Laura Morello
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  2. Mauro Bardini
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  3. Mauro Cricrì
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  4. Francesco Sala
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  5. Diego Breviario
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Corresponding author

Correspondence to Laura Morello.

Additional information

GenBank accession number for OsCDPK2 cDNA: X81394; OsCDPK 5’flanking region:Y13658

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Morello, L., Bardini, M., Cricrì, M. et al. Functional analysis of DNA sequences controlling the expression of the rice OsCDPK2 gene. Planta 223, 479–491 (2006). https://doi.org/10.1007/s00425-005-0105-z

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