Genomics, molecular genetics & biotechnology: A rapid and direct approach to identify promoters that confer high levels of gene expression in monocots
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abstract
Direct screening of genomic libraries for highly expressed genes is an efficient way to identify promoters that confer high levels of gene expression. To test the efficacy of this approach to isolate promoters for directing high levels of gene expression in sugarcane, 11 genomic clones were isolated after screening a sugarcane genomic library with radioactively labeled first strand cDNA probes synthesized from sugarcane leaf mRNA. Hybridization analysis with pooled first strand cDNA indicated that eight of the clones probably contain genes with expression levels similar to or higher than ubiquitin (which was also isolated in this screen). The coding regions of these 11 genomic clones were isolated from a cDNA library, sequenced, and found to represent parts of five different genes including elongation factor 1, -tubulin, an aquaporin, a proline-rich protein, and one novel gene. Southern and Northern results showed that the sugarcane proline-rich protein-encoding gene (SPRP1) was the best candidate to isolate a promoter that would direct high levels of expression, although the other four are also good candidates. Two genomic clones of the sugarcane proline-rich gene and a sugarcane elongation factor (SEF1) gene, which contain both the promoter and coding regions, were subcloned, sequenced, and promoter regions defined by comparison of cDNA and genomic DNA sequences. Both the SEF1 and SPRP1 promoter/ -glucuronidase (GUS) fusions resulted in high levels of GUS expression when reintroduced into embryogenic sugarcane callus, and also resulted in high levels of transient GUS expression in wheat embryos. This approach has value as an efficient technique to find promoters that confer high-level gene expression in monocots.
