In this study, we identified a potential bidirectional promoter from the genome of Brassica napus (Fig. 1a). Through the cloning and functional analysis of the promoter PBn265 of Brassica napus, we have drawn the following preliminary conclusions: the promoter has transcriptional activity in both two directions (Fig. 1b,c); the promoter may be a bidirectional promoter, containing a variety of cis-acting elements related to light response (Fig. 2); the PBn265-F has activity in the root, stem, leaf, flower and silique in transgenic Arabidopsis thaliana (Fig. 4), and that SHH2 may also express in all tissues in Brassica napus. On the other hand, the expression level of PBn265-R was relatively high in green tissues such as stem and leaf, but significantly lower or not even present in the roots. In stem and leaf tissues, the expression level of EGFP and GUS decreased significantly in the absence of light. After resumption of light, the gene expression level rose gradually, and with the prolonging of the light, the gene expression level increased too. Similar patterns were not observed in the root. Therefore, we hypothesized that CFM3 is mainly expressed in chloroplasts, as there are fewer chloroplasts in roots. Based on these results, when PBn265 simultaneously drives the expression of both two genes, PBn265-R may affect the function of PBn265-F (Fig. 5).
The leaf is the photosynthesis organ of the plant, playing an important role in energy fixation and utilization. The study of light-induced promoters has important theoretical significance and application value for the further study of genes for which light regulates expression . Light-induced promoters with bidirectional driving function have a potentially broad application in plant genetic improvement and genetic engineering. In seven species of Brassicaceae, the arrangement of the structure, “CFM3-bidirectional promoter-SHH2”, has a high degree of conservation of synteny (Supplementary Table. S2). This evolutionarily conservative arrangement may have some unknown biological significance and these promoters may all be bidirectional promoters. Two genes that share a short promoter can potentially save on resource usage, such as time and space, energy, and RNA polymerase. This is highly conducive to an organism's growth and development. Therefore, the evolutionary origin of this promoter deserves further study.
It is worth noting that, in previous studies on bidirectional promoters, only 9% of them have TATA-box . It was speculated that the regulation mechanism of bidirectional promoters without TATA-box may be different from directional promoters . However, this short (265bp) sequence contains two TATA-box and rich CpG islands, but low GC content. Among the promoters randomly selected from the human genome, the average GC content is 53% and about 70.8% of them have GC content higher than 60%, significantly higher than average . We found that PBn265 has only 42% GC content and does not contain CpG islands (Supplementary Fig. S1). This may be a new discovery of the characteristics of bidirectional promoters, indicating that there may be new regulation mechanisms for bidirectional promoters, which will help future generations to further study the regulatory mechanism of bidirectional promoters.
In summary, the light-induced bidirectional promoter found in this study is the shortest among the natural inducible bidirectional promoters as far as we know, and it also has some characteristics different from other promoters, containing two TATA-boxes, having low GC content and lacking CpG islands. In addition, considering the promotion activity and the applicability in heterologous species, the promoter has broad application potential in plant genetic improvement and genetic engineering.