Isolation and Functional Characterization of Bidirectional Promoters in Rice

Abstract

Bidirectional promoters, which show great application potential in genetic improvement of plants, have aroused great research interest recently. However, most bidirectional promoters were cloned individually in the studies of single genes. Here, we initiatively combined RNA-seq data and cDNA microarray data to discover the potential bidirectional promoters in rice genome. Based on the expression level and correlation of each adjacent and oppositely transcribed gene pair, we selected four candidate gene pairs. Then, the intergenic region between each pair was isolated and cloned into a dual reporter vector pDX2181 for functional identification. GUS and GFP assays of the transgenic plants indicated that all the intergenic regions showed bidirectional expression activity in various tissues. Through 5' and 3' deletion analysis on one of the above bidirectional promoters, we identified the enhancing region which sharply increased its bidirectional expression efficiency and the essential regions respectively responsible for its 5' and 3' basic expression activity. The bidirectional arrangement of the four gene pairs in six gramineous plants was also analyzed, showing the conserved characteristics of the four bidirectional promoters identified in our study. In addition, two novel cis-sequences conserved in the four bidirectional promoters were discovered by bioinformatic identification. Our study proposes a feasible method for selecting, cloning, and functionally identifying bidirectional promoters as well as for discovering their bidirectional regulatory regions and conserved sequences in rice.

Keywords: GFP assay; GUS assay; bidirectional promoter; conservation analysis; deletion analysis; rice; stable transformation.

Figure 1

Schemes of constructs carrying BIP1 and different deleted versions fused with GFP and GUS reporter genes.

Figure 2

Histological analysis of GFP and GUS expression in various tissues of the transgenic plants containing different GFP/bidirectional promoter/GUS fusions. (A–D), plants containing GFP::BIP1-BIP4::GUS. Localization of GFP is shown at the left area; Localization of GUS is shown at the right area.

Figure 3

Quantitative analysis of GFP and GUS expression in various tissues of the transgenic plants containing different GFP/bidirectional promoter/GUS fusions. (A–D), plants containing GFP::BIP1-BIP4::GUS. a, b, c, d, e: significant difference (P < 0.05). Error bars indicate _SE based on five independent biological replicates.

Figure 4

Quantitative analysis of GFP and GUS expression of the BIPs transgenic plants in response to melatonin. (A–D), plants containing GFP::BIP1-BIP4::GUS. a: no significant difference. Error bars indicate _SE based on five independent biological replicates.

Figure 5

Histological analysis of GFP and GUS expression in various tissues of the transgenic plants containing different GFP/BIP1 deleted version/GUS fusions. Localization of GFP is shown at the left area; Localization of GUS is shown at the right area. R1 (blue dashed line), region 1; R2 (red dashed line), region 2; R3 (green dashed line), region 3.

Figure 6

Quantitative analysis of GFP and GUS expression in various tissues of the transgenic plants containing different GFP/BIP1 deleted version/GUS fusions. 1F2R, plants containing GFP::BIP1-1F2R::GUS; 1F3R, 2F1R and 2F2R follow the same pattern. a, b, c, d: significant difference (P < 0.05). Error bars indicate _SE based on five independent biological replicates.

Figure 7

Two novel cis-sequences conserved in the four identified bidirectional promoters. The frequencies of the two cis-sequences in the intergenic regions between divergent gene pairs in rice genome and in the random promoters used as a reference set are shown on the second column and the third column, respectively.