Abstract
As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.
Highlights
Bacterial pathogens or phytohormone treatments demonstrated that inducible synthetic promoters can function in transgenic tobacco and Arabidopsis[5]
If a single cis-element is added in the combinations, it should be quadrupled to increase its effect and placed upstream of a promoter, which is better to be a minimal promoter to avoid the interference from numerous cis-elements[5,17]
We fused several regulatory sequences related to tissue-specific expression in 8 different combinations
Summary
Bacterial pathogens or phytohormone treatments demonstrated that inducible synthetic promoters can function in transgenic tobacco and Arabidopsis[5]. This study proves that the new high-throughput screening method has a high application potential in the discovery of inducible cis-elements. A high-throughput method for screening and identifying cis-elements related to tissue-specific expression is critical to the development of tissue-specific synthetic promoters. As the feasibility of synthesizing tissue-specific promoters in rice was proved, an available method for the discovery of tissue-specific cis-elements is important to the development of tissue-specific synthetic promoters. We identified a general regulatory sequence 5′-AAAATATTTAT-3′ (the underlined sequence indicates the core element), which can be applied in the synthesis of green tissue-specific promoters. Our study provides an example for developing tissue-specific synthetic promoters in rice, and proposes a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice
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