|Meagher, Richard -|
Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 17, 2009
Publication Date: January 29, 2010
Citation: An, Y., Meagher, R.B. 2010. The Strong Expression and Conserved Regulation of ACT2 in Arabidopsis and Physcomitrella Patens. Plant Molecular Biology Reporter. 28:481-490. Interpretive Summary: Plant research and crop improvement strategies often require strong gene promoters, sequences that regulate the expression of a specific gene, to produce high levels of desirable gene products in transgenic plants. In this study, we demonstrate that ACT2 5’ regulatory region (the promoter for the Actin 2 gene) was able to generate more gene product, i.e., was more active, than one of most commonly used promoters, the CaMV 35S promoter. The strong activity of the ACT2 5’ regulatory region was observed in evolutionarily distant plant species including Arabidopsis and moss cells. Thus, this regulatory region can serve as a strong regulator for foreign gene expression in a wide range of plant species and crops. We also identified several conserved sequences and structures, by sequence comparison across ACT2 genes from different species (evolutionary footprints), in the ACT2 5’ regulatory region and validated their importance by expression analysis in divergent plant species. Some of these evolutionary footprints play a role in the regulation of ACT2 expression suggesting that evolutionary footprinting and across species validation can provide an effective approach to identify key regulatory components within plant promoters. The study demonstrates that the validation of evolutionary footprints can make the identification of novel regulatory elements for plant research and crop improvement more efficient and thus improve our ability to perform crop improvement through biotechnology.
Technical Abstract: Actin genes encode structurally and functionally conserved proteins essential in a variety of cellular functions across eukaryotic species. In this report, we demonstrated that regulatory activity of ACT2 5’ regulatory region was significantly higher than that of the CaMV 35S promoter in Arabidopsis and Physcomitrella patens. The activity of the ACT2 5’ regulatory region was also observed in rice and maize tissues. Thus it should be able to serve as a strong regulator to express foreign genes in divergent plant species. Comparing with other plant actin genes, we observed several conserved features in ACT2 5’ regulatory region: 1) a dozen sequence elements identical between ACT2 and ACT8 5’ regulatory regions. 2) An intron at 5’ untranslated regions (UTR) of all characterized plant actin genes. 3) A pyrimidine rich region following a purine rich region (PuPy) at 5’ UTRs of several plant actin genes. Gene regulation is a product of evolution. Preservation of such conserved features (evolutionary footprints) in the less constrained sequence region suggests that they may play roles in gene regulation. Site mutagenesis and transient expression studies showed that some of the conserved sequence elements indeed played roles in gene regulation of ACT2 in Arabidopsis. The PuPy sequence region and splicing of the intron were required for its strong expression in Arabidopsis. Interestingly, the intron enhancing expression mechanism was conserved in Physcomitrella patens. Therefore, preservation of the conserved sequence elements and structures are partly due to its regulatory importance.