Abstract
The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5′UTR sequence is interrupted by a 446 bp long leader intron. This feature is shared with two other rice β-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant β-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general picture is drawn for three rice α-tubulin and two rice β-tubulin genes in which the first intron of the coding sequence for the formers and the intron present in the 5′UTR for the latters, are important elements for controlling gene expression. We used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants. The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect.
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Abbreviations
- IME:
-
Intron-mediated enhancement
- IDSE:
-
Intron Dependent Spatial Expression
- GUS:
-
Beta-glucuronidase
- TSS:
-
Transcription start site
- UTR:
-
Untranslated region
- MT:
-
Microtubule
- bp:
-
Base pairs
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We deeply thank Prof. Francesco Sala, University of Milan, for providing the required greenhouse facilities.
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Supplementary Figure 1
Diagrams of the different genomic organization of the rice α- and β-tubulin genes and related constructs for plant transformation. Top: the two diagrams summarize the different organization of typical α- and β-tubulin genes with respect to the position of the regulatory intron (Intron I for α-tubulin and Leader I for β-tubulin). R: regulatory sequences e.g. promoters and introns. TSS and ATG mark the 5’end of the transcript and the translation initiation codon, respectively. Variability in promoter and intron length is also shown. The dashed boxes indicate further downstream indefinite sequences. Bottom: The six rice tubulin:GUS fusion constructs used in plant transformation. The GUS coding region is represented by an open arrow which indicates the direction of transcription. Ter indicates the nopaline synthase terminator. LI: leader intron (TIFF 65 kb)
Supplementary Figure 2
Northern blot analysis of tubulin α2 and tubulin α3 mRNA levels in different rice organs. Total RNA was extracted from the indicated organs and aliquots were hybridized with α2- or α3- tubulin isotype-specific probes (TIFF 98 kb)
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Gianì, S., Altana, A., Campanoni, P. et al. In trangenic rice, α- and β-tubulin regulatory sequences control GUS amount and distribution through intron mediated enhancement and intron dependent spatial expression. Transgenic Res 18, 151–162 (2009). https://doi.org/10.1007/s11248-008-9202-7
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DOI: https://doi.org/10.1007/s11248-008-9202-7