Isolation and Characterization of a Green-Tissue Promoter from Common Wild Rice (Oryza rufipogon Griff.)

Abstract

Promoters play a very important role in the initiation and regulation of gene transcription. Green-tissue promoter is of great significance to the development of genetically modified crops. Based on RNA-seq data and RT-PCR expression analysis, this study screened a gene, OrGSE (GREEN SPECIAL EXPRESS), which is expressed specifically in green tissues. The study also isolated the promoter of the OrGSE gene (OrGSEp), and predicted many cis-acting elements, such as the CAAT-Box and TATA-Box, and light-responding elements, including circadian, G-BOX and GT1 CONSENSUS. Histochemical analysis and quantification of GUS activity in transgenic Arabidopsis thaliana plants expressing GUS under the control of OrGSEp revealed that this promoter is not only green tissue-specific, but also light-inducible. The ability of a series of 5’-deletion fragments of OrGSEp to drive GUS expression in Arabidopsis was also evaluated. We found that the promoter region from −54 to −114 is critical for the promoter function, and the region from −374 to −114 may contain core cis-elements involved in light response. In transgenic rice expressing GUS under the control of OrGSEp, visualization and quantification of GUS activity showed that GUS was preferentially expressed in green tissues and not in endosperm. OrGSEp is a useful regulatory element for breeding pest-resistant crops.

Keywords: Green tissue-specific expression; Promoter; common wild rice; light-induced.

Figure 1

OrGSE gene expression in roots and shoots of common wild rice. The mRNA levels of OrGSE gene were determined in different tissues and developmental stages by RT-PCR with 26, 30 and 34 cycles of amplification. (a) The gel figure of RT-PCR. The rice Actin gene was used as an internal control. SR: Shooting stage Root, SL: Shooting stage Leaf, HR: Heading stage Root, HL: Heading stage Leaf, and HS: Heading stage Stem; (b) qPCR analysis of transcript levels of OrGSE in different tissues. Data are the means of three replicates, and error bars show the standard error.

Figure 2

The location of putative cis-acting elements in OrGSEp-374 predicted by the PlantCARE database and schematic diagrams of promoter deletion constructs. (a) Putative cis-acting elements in OrGSEp-374, The 5′-region of the OrGSE gene containing the 374 bp promoter sequence and 166 bp sequence downstream of the translational start site. The transcription initiation site is defined as +1. The TATA box, CAAT box and other key cis-acting elements are underlined with and indicated by different colors as shown in the legend. The position of each element is also indicated by schematic diagrams; (b) The schematic diagrams of the truncated OsGSE-374 constructs. The numbers to the left of these diagrams indicate the position of the 5′-deletion.

Figure 3

GUS histochemical assays in transgenic Arabidopsis T3 seedlings. GUS histochemical assays in transgenic Arabidopsis T3 seedlings harboring constructs with GUS expression driven by the CaMV 35S promoter (35S: GUS), OrGSEp-374 (OrGSEp-374) and different 5′-deletion fragments (OrGSEp-274, OrGSEp-204, OrGSEp-114 and OrGSEp-54), during vegetative growth. Photographs were taken 3 days, 5 days and 14 days after seed germination.Bar = 1 cm.

Figure 4

GUS staining in siliques, leaves and roots sampled during the reproductive stage from transgenic T3 Arabidopsis seedlings carrying OrGSEp-374, OrGSEp-274, OrGSEp-204, OrGSEp-114 and OrGSEp-54.Bar = 1 cm.

Figure 5

Quantification of GUS activity in transgenic T3 Arabidopsis roots and shoots carrying the CaMV 35S promoter, OrGSEp-374 and different 5′-deletion fragments constructs. Gus activity measurements are shown for three positive transgenic lines for each OrGSEp promoter construct. CaMV35S: GUS is an independent transgenic plant carrying the CaMV35S promoter construct. OrGSEp-374#3, OrGSEp-374#8 and OrGSEp-374#12 are three positive transgenic lines carrying the OrGSEp-374 construct. OrGSEp-274#4, OrGSEp-274#9 and OrGSEp-274#15 are three independent transgenic lines carrying the OrGSEp-274 construct. OrGSEp-204#1, OrGSEp-204#5 and OrGSEp-204#7 are three positive lines carrying the OrGSEp-204 construct. OrGSEp-114#2, OrGSEp-114#8 and OrGSEp-114#9 are three lines carrying the OrGSEp-114 construct. OrGSEp-54#3, OrGSEp-54#6 and OrGSEp-54#11 are three transgenic lines carrying the OrGSEp-54 fragment. Data are the means of three replicates, and error bars show the standard error. The “**” indicates that a significant difference (p < 0.001) in GUS activity was detected between root and shoots of plants carrying the same promoter construct.

Figure 6

GUS staining and quantification of GUS activity of transgenic Arabidopsis seedlings containing different 5’-promoter deletion fragments grown under light and dark conditions. (a) Ten-day-old seedlings were stained to observe GUS expression. Bar = 1 cm; (b) Quantification of GUS activity in 20-day-old T3 transgenic Arabidopsis grown under light and dark conditions. OrGSEp-374#3, OrGSEp-374#8 and OrGSEp-374#12 are three positive transgenic lines carrying the OrGSEp-374 construct. OrGSEp-274#4, OrGSEp-274#9 and OrGSEp-274#15 are three independent transgenic lines carrying the OrGSEp-274 construct. OrGSEp-204#1, OrGSEp-204#5 and OrGSEp-204#7 are three positive lines carrying the OrGSEp-204 construct. OrGSEp-114#2, OrGSEp-114#8 and OrGSEp-114#9 are three lines carrying the OrGSEp-114 construct. Data are the means of three replicates, and standard errors are shown by error bars. The “**” indicates that a significant difference (p < 0.001) in GUS activity was detected between roots and shoots of seedlings carrying the same promoter construct.

Figure 7

GUS histochemical assays and GUS activity in different tissues of OrGSEp-374 transgenic rice. (a) GUS histochemical assays of OrGSEp-374 transgenic rice. A. Root, B. stem, C. leaf, D. ligule, E. spikelet, F. anther, G. seed. H. endosperm. Bar = 1000 μm; (b) GUS activity in different tissues of transgenic rice carrying the OrGSEp-374 construct. Boxplots show GUS activity in different tissues of transgenic rice. The lower boundary of each box denotes the 25th percentile, the upper boundary of each box denotes the 75th percentile, and the solid line in the middle of each box denotes the 50th percentile. The two ends of the error bars denote the maximum and minimum values.