Two Novel Positive cis-Regulatory Elements Involved in Green Tissue-Specific Promoter Activity in Rice (Oryza sativa L ssp.)

In plant genetic engineering, use tissue-specific promoters to control the target genes’ expression is an effective way to avoid the adverse effects caused by constitutive promoters. However, tissue-specific promoters with strong, reliable expression that are used for various crop improvements are limited. Based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120), and utilized the GUS reporter gene to demonstrate that the DX1 gene promoter (P_DX1) is a green tissue-specific promoter in transgenic rice. To identify the regulatory mechanism controlling the expression of the DX1 gene, we performed a series of 5’ and 3’ deletions, gel mobility shift assays and site-directed mutagenesis studies, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P_DX1. GSE1 acts as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acts as a positive regulator only in sheath and stem tissue, and has a weaker effect on gene expression. Quantitative analyses of GUS activity indicate that P_DX1:GUS is highly expressed in green tissues. In addition, P_DX1:GUS is not expressed in anther and seed, and this characteristic reduces the potential ecological risk and potential food safety issues. Identification of the promoter and its cis regulatory elements will prove useful to the field of rice transgenic breeding.