High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots

Abstract

Expression patterns of a rice (Oryza sativa) cytochrome c gene OsCc1 and its promoter activity were characterized in transgenic rice plants. OsCc1 transcripts accumulate in most cell types, but to varying levels. Large amounts of OsCc1 transcripts are found in the roots, calli, and suspension cells, but relatively lower in mature leaves, demonstrating its higher levels of expression in non-photosynthetic tissues. Unlike the human cytochrome c gene, which is responsive to cAMP, OsCc1 expression is not enhanced in various rice tissues after dibutyryl cAMP treatments. OsCc1 promoter was linked to the sgfp gene and its activities in different tissues and cell types of transgenic rice plants were analyzed in comparison with the Act1 and RbcS promoters. OsCc1 promoter directs expression in virtually all organs of transgenic plants including roots, leaves, calli, embryos, and suspension cells, showing a particularly high activity in calli and roots. Activity of the OsCc1 promoter was 3-fold higher than Act1 in calli and roots and comparable with RbcS in leaves, representing a useful alternative to the maize (Zea mays) Ubi1 and the rice Act1 promoters for transgene expression in monocots.

Figure 1

A, Physical map of the genomic clone (OsCc1) encoding a rice cytochrome c. The solid boxes represent exons. A partial cDNA (cyt) that was used as a probe for RNA-blot experiments is indicated. The transcriptional start site and the translational start codon are marked with +1 and ATG, respectively. Sequences that are similar to those of CRE and NRF1-binding sites are indicated by I and II, respectively. Important restriction enzyme sites are abbreviated as follows: C, ClaI; RI, EcoRI; S, SalI; and X, XbaI. B, The expression of OsCc1 in different tissues of rice. RNA-blot analysis was carried out using total RNA from rice suspension cells (SC), leaves (L), roots (R), and calli (C). C, The response of OsCc1 mRNA expression to the Bt₂cAMP treatment. Suspension cells (SC), leaves (L), and roots (R) were treated with 1 mm dibutyryl cAMP (Bt₂cAMP) for the time periods indicated, and total RNA was extracted. Total RNA was fractionated on a denaturing agarose gel, blotted to a nylon membrane, and hybridized with a [³²P]-labeled cyt cDNA probe, shown in A (OsCc1). The loading of an equal amount of total RNA in each lane was verified by ethidium bromide staining (EtBr).

Figure 2

The response of OsCc1 mRNA expression in etiolated seedlings to light exposure. Etiolated seedlings were treated with light at 150 μmol m² s⁻¹ for the time periods indicated, and total RNA was extracted. Total RNA was fractionated on a denaturing agarose gel, blotted to a nylon membrane, and hybridized with either a [³²P]-labeled cyt cDNA probe, shown in Figure 1A (OsCc1), or the coding region for a small subunit of ribulose-1,5-bisphosphate carboxylase (RbcS). The loading of an equal amount of total RNA in each lane was confirmed by ethidium bromide staining (EtBr). Lane L shows total RNA extracted from mature leaves.

Figure 3

The expression vectors used for rice transformation. pSB-CG (OsCc1::sgfp) consists of the OsCc1 promoter linked to the sgfp coding region, the 3′ region of the potato proteinase inhibitor II gene (3′pinII), and the bar gene expression cassette that contains a 35S promoter/bar coding region/3′ region of the nopaline synthase gene (3′nos). pSBG700 (Act1::sgfp) and pSB-RG (RbcS::sgfp) are identical to pSB-CG except that the rice Act1 and RbcS promoters, respectively, are fused to the sgfp-coding region. Important restriction sites are indicated: EcoRV (E), NcoI (N), XbaI (X), BamHI (B), and SpeI (S). Restriction enzymes followed by the expected fragments and hybridization probe (probe) used for genomic DNA-blot analyses are shown below the map.

Figure 4

Genomic DNA-blot analysis of transgenic rice plants. A, Genomic DNAs from the leaf tissues of three (1–3) independent lines of OsCc1::sgfp-transformed plants were digested with NcoI (N), EcoRV (E), and hybridized with a 0.7-kb DNA fragment containing the sgfp coding region (see Fig. 3). PC contains NcoI-digested pSB-CG. B, Genomic DNAs from the leaf tissues of three (1–3) independent lines of Act1::sgfp-transformed plants were digested with BamHI (B), XbaI (X), and hybridized with the same probe described in A. PC contains BamHI-digested pSBG700. C, Genomic DNAs from the leaf tissues of three (1–3) independent lines of RbcS::sgfp-transformed plants were digested with XbaI (X), SpeI (S), and hybridized with the same probe described in A. PC contains XbaI-digested pSB-RG. NC, Genomic DNAs from an untransformed control plant; 1X, 3X, and 5X in PC represent one, three, and five genome equivalents of pSB-CG (A), pSBG700 (B), or pSB-RG (C), relative to 5 μg of rice genomic DNA, respectively. The DNA molecular size markers (M) are indicated.

Figure 5

Images of sGFP fluorescence in transgenic rice plants expressing OsCc1::sgfp (OsCc1), Act1::sgfp (Act1), and RbcS::sgfp (RbcS) chimeric genes. A, sGFP fluorescence in transgenic rice seedlings (left) and uncoated dry seeds (right) taken by a digital video imaging system. NT represents an untransformed rice seedling (right) and seeds (left). B, Confocal microscopic image of sGFP fluorescence in a leaf, a root apex, elongating region of a root, and secondary root of transgenic rice plants. GC, Guard cells; RA, root apex; RC, root cap; RHP, root hair protuberances; LC, long cells; SC, short cells; PR, primary root; SR, secondary root. Scale bars = 30 μm.

Figure 6

RNA gel-blot analysis and relative levels of sgfp transcripts in transgenic rice plants shown in Figure 4. A, Total RNAs extracted from leaf, root and callus of three (1–3) independent lines of OsCc1::sgfp-, Act1::sgfp-, and RbcS::sgfp-transformed plants and from an untransformed control plant (NT) were hybridized with a 0.7-kb DNA fragment containing the sgfp coding region (see Fig. 3). Hybridizations with the rice RL5 encoding the 5S rRNA-binding protein (Kim and Wu, 1993) were used for equal RNA loading. B, Transcript levels of sgfp shown in A were calculated using those of corresponding RL5 as a reference and the resultant values were then normalized to 1 for that from leaf tissues of Act1::sgfp-transgenic line 1.