Plant Actin promoters
In the quest to improve the expression of foreign genes in transformed cereals, several promoter regions originating from monocot plants have been tested. In 1990, McElroy and colleagues from Cornell University reported that the 5′ region of the rice actin 1 (Act-1) gene successfully directed the expression of a reporter gene in transformed rice protoplasts. Since then, the promoter of the rice Act-1 gene has been used as a strong constitutive promoter driving the expression of genes of interest in monocots.
Actin is a fundamental component of the cell cytoskeleton present in all tissues. Cell shape determination, cell division, organelle movement and extension growth are all believed to involve actin proteins.
The Act-1 gene from rice has a short 5′ non-coding exon, separated by a 447 bp intron (intron 1) from the first coding exon. The presence of the first intron of the gene has proved to be fundamental for the efficient gene expression from the Act-1 promoter. The region is active in almost all plant tissues except sporophytic and gametophytic pollen tissues.
All the necessary cis-acting regulatory elements for the activity of the Act-1 promoter are in the region 834 bp upstream of the transcription start site. Apart from the TATA box, there are two regions that play a role in the regulation of the constitutive expression of the promoter. A poly (dA-dT) element of 38 bp between -245 and -152 position is the binding sequence for a trans-acting protein that works as a positive regulator of Act-1 promoter activity. A region between -300 and -260 positions contains CCCAA pentamer repeats that appear to be involved in negative regulation of promoter activity. Its action seems to be tissue-specific, particularly in roots.
The portion of the rice Act-1 gene used in vectors for monocot transformation normally contains:
- approximately 1 kb of regulatory sequences located 5′ of the transcribedregion,
- the 5′ non-coding exon 1,
- the intron 1, and
- the coding exon 2 of the Act-1 gene.
The regulatory region of rice Act-1 gene has been successfully used for expressing diverse genes of interest after transformation of cereals, i.e. maize, rice, barley, wheat and rice.
1. Promoter region derived from the rice Act-1 gene
The rice Act-1 promoter region is protected under a U.S. patent granted to the Cornell Research Foundation, Inc. In the description of the invention, the definition of a promoter regulatory region includes more sequence than the minimal elements of a promoter region: TATA box, AGGA box and transcription start site. The claimed promoter region encompasses sequences located 5′ to the translational start site that are essential for conferring a high level of expression of the gene under its control.