Isolation and Functional Characterization of a Green-Tissue Promoter in Japonica Rice (Oryza sativa subsp. Japonica)

Abstract

Plant promoters play a vital role in the initiation and regulation of gene transcription. In this study, a rice protein/gene of unknown expression, named Os8GSX7, was gained from a rice T-DNA capture line. The semi-quantitative RT-PCR analysis showed that the gene was only expressed in root, glume, and flower, but not in stem, leaf, embryo, and endosperm of japonica rice. The GUS activity analysis of the GSX7R promoter showed that it was a reverse green tissue expression promoter, except in endosperm. The forward promoter of GSX7 cannot normally drive the expression of the foreign GUS gene, while the reverse promoter of GSX7 is a green tissue-specific expression promoter, which can drive the expression of the foreign GUS gene. The region from -2097 to -1543 bp was the key region for controlling the green tissue-specific expression. The regulatory sequences with different lengths from the 2097 bp reverse sequence from the upstream region of the Os8GSX7 were fused with the GUS reporter gene and stably expressed in rice. Furthermore, transgenic rice plants carrying Cry1Ab encoding Bacillus thuringiensis endotoxin, regulated by GSX7R, were resistant to yellow stem borer. The analysis suggested that 10 light responsive elements of tissue-specific expression were found, including ACE, Box4, CAT-box, G-Box, G-box, GATA motif, GC motif, I-box, Sp1, and chs-unit1 M1. In addition, the results of 5' and 3' deletions further speculated that ACE and I-box may be the key elements for determining the green tissue-specific expression of GSX7R promoter.

Keywords: RT-PCR; agrobacterium; cloning; green-tissue specific expression; promoter.

Figure 1

A schematic diagram showing the insertion position of T-DNA.

Figure 2

Schematic diagram of the T-DNA region of binary vector pCXGUS-P with GSX7R or GSX7R deletions.

Figure 3

Expression profiles of Os8GSX7 and GUS by RT-PCR analysis. (A) Expression profiles of Os8GSX7 gene by RT-PCR analysis in the wild type; (B) Expression profiles of GUS gene in transgenic positive lines of GSX7R-2097 by RT-PCR analysis. The rice Actin gene was used as an internal control.

Figure 4

The PCR amplification of promoter GSX7R and its deletions. M: M5 DL2000 plus DNA marker, 1: GSX7R-2097, 2: GSX7R-1765, 3: GSX7R-1198, 4: GSX7R-554, 5: GSX7R-1543, 6: GSX7R-1212. The amplified bands are indicated by red arrows.

Figure 5

The location of putative cis-acting elements in GSX7R predicted by the PlantCARE database and schematic diagrams of promoter deletion constructs. (a) Putative cis-acting elements in GSX7R, The reverse 5′-region of the Os8GSX7 gene containing the 2097 bp promoter sequence from the translational start site. The transcription initiation site is defined as +1. The GC-motif, G-box, and other key cis-acting elements are underlined with and indicated by black frame as shown in the legend and different colors outside. The position of each element is also indicated by schematic diagrams; (b) The schematic diagrams of the truncated GSX7R constructs. The numbers to the left of these diagrams indicate the position of the 5′-deletion or 3′-deletion.

Figure 6

PCR analysis of hpt in different transgenic rice. M: M5 Marker II DNA Marker, 1–2: GSX7R-2097; 3–4: GSX7R-1765, 5–6: GSX7R-1198, 7–8: GSX7R-554, 9–10: GSX7R-1543, 11–12: GSX7R-1212, 13: non-transgenic rice, 14: Positive plasmid control.

Figure 7

GUS staining and quantification of GUS mRNA transcript of transgenic rice containing different 5′ and 3′-deletion. (a) Histochemical analysis of transgenic rice plants containing various GSX7R promoter fragment/GUS fusions in different tissues; (b) GUS mRNA expression in the leaf of various rice transformants. Quantitative qRT-PCR analysis of GUS transcripts from the leaf in transgenic rice plants. Data are shown as mean ± SD (n = 3), Student’s t-test. *** indicates highly significant differences from all tested tissues for each transgenic plant. Relative expression was calculated as lg₂^−ΔΔCt.

Figure 8

Schematic diagram of vector construction and bioassays in the field. (a) GSX7R:Cry1Ab:p1300GSAb plant transformation vector containing the Cry1Ab gene driven by the GSX7R promoter; (b) (A,C) Transgenic plants without any symptom of infestation in the whole-plant bioassay; (B,D) Wild-type plants showed white heads in the whole-plant bioassay. Red arrow indicates damage to YSB.