A Rapid and Direct Approach to Identify Promoters That Confer High Levels of Gene Expression in Monocots

specific hybridization has reduced disease problems, increased biomass yield and sugar yield, and improved Direct screening of genomic libraries for highly expressed genes adaptability for growth under various stress conditions is an efficient way to identify promoters that confer high levels of (Roach, 1972; Srivastava et al., 1994). gene expression. To test the efficacy of this approach to isolate promoters for directing high levels of gene expression in sugarcane, 11 The complicated genetics and long breeding cycle for genomic clones were isolated after screening a sugarcane genomic sugarcane make transformation an attractive approach library with radioactively labeled first strand cDNA probes syntheto include in the toolbox for sugarcane improvement. sized from sugarcane leaf mRNA. Hybridization analysis with pooled The most commonly used method for transformation first strand cDNA indicated that eight of the clones probably contain of sugarcane is particle bombardment of embryogenic genes with expression levels similar to or higher than ubiquitin (which callus combined with a herbicide resistance gene as a was also isolated in this screen). The coding regions of these 11 genoselectable marker (Gallo-Meagher and Irvine, 1993, mic clones were isolated from a cDNA library, sequenced, and found 1996; Bower and Birch, 1992; Bower et al., 1996). Proto represent parts of five different genes including elongation factor duction of transgenic sugarcane plants by intact cell 1 , -tubulin, an aquaporin, a proline-rich protein, and one novel gene. electroporation (Arencibia et al., 1995) and by AgroSouthern and Northern results showed that the sugarcane proline-rich protein-encoding gene (SPRP1) was the best candidate to isolate a bacterium-mediated transformation of embryogenic calpromoter that would direct high levels of expression, although the lus has also been reported (Enriquez-Obregon et al., other four are also good candidates. Two genomic clones of the sugar1998; Arencibia et al., 1998). cane proline-rich gene and a sugarcane elongation factor 1 (SEF1 ) In conjunction with an efficient transformation sysgene, which contain both the promoter and coding regions, were tem, the availability of a range of promoters with varied subcloned, sequenced, and promoter regions defined by comparison strengths and tissue specificities is critical to the success of cDNA and genomic DNA sequences. Both the SEF1 and SPRP1 of transgenic approaches to crop improvement. The promoter/ -glucuronidase (GUS) fusions resulted in high levels of CaMV 35S promoter has been widely used for highGUS expression when reintroduced into embryogenic sugarcane callevel constitutive expression in dicots, but confers lower lus, and also resulted in high levels of transient GUS expression in wheat embryos. This approach has value as an efficient technique to levels of expression in monocots. Promoters isolated find promoters that confer high-level gene expression in monocots. from monocots generally show higher activity in monocots, and adding an intron between the promoter and the reporter gene can increase transcription levels (Wilmink et al., 1995; Rathus et al., 1993; Maas et al., 1991; S is one of the world’s most important crops, with the 2001–2002 world cane sugar crop forecast Callis et al., 1987). The rice actin promoter Act1 (McElat a near record 126.8 million megagrams (FAS, 2001). roy et al., 1990, 1991; Wang et al., 1992; Zhang et al., Modern cultivated sugarcane is believed to be derived 1991) and the maize ubiquitin promoter Ubi (Chrisfrom interspecific hybrids between the domesticated tensen and Quail (1996)) achieved far better expression species Saccharum officinarum L. and its wild relative than the CaMV 35S in most monocots tested. Among S. spontaneum L. Chromosome numbers of sugarcane promoters tested in sugarcane, the Emu promoter and cultivars range from 100 to 130 with approximately 10% the maize ubiquitin promoter showed higher levels of derived from S. spontaneum (Simmonds, 1976). Interexpression than the CaMV 35S promoter (McElroy et al., 1991; Gallo-Meagher and Irvine, 1993; Rathus et al., 1993), curiously the opposite of their relative strengths Meizhu Yang and Mark D. Burow, Department of Soil and Crop in dicots (Callis et al., 1990; Mitra and Higgins, 1994). Sciences, Texas A&M University, College Station, TX 77843; Robert Variation in transgene expression levels between differBower, Grain Biotech Australia, Perth, Western Australia; Andrew H. Paterson, Plant Genome Mapping Laboratory, University of Georgia, ent species and promoters may be due to different abunRm. 162, Riverbend Research Center, 110 Riverbend Rd., Athens, dance of transcription factors, recognition of promoter GA 30602; T. Erik Mirkov, Department of Plant Pathology and Microsequences or intron splicing sites (Wilmink et al., 1995) biology, Texas A&M University Agricultural Experiment Station, or other factors. 2415 East Highway 83, Weslaco, TX 78596. Received 17 June 2002. Sugarcane, as well as other monocots, may be a useful *Corresponding authors (Paterson: [email protected]. edu; Mirkov: [email protected]). source of promoters that can provide specific transgene expression patterns in valuable monocot crop species, Published in Crop Sci. 43:1805–1813 (2003). but there are few published results on sugarcane pro Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA moters (Wei et al., 1999). We seek to begin to fill this