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

^a Department of Soil and Crop Sciences, Texas A&M University, College Station, TX 77843
^b Grain Biotech Australia, Perth, Western Australia
^c Plant Genome Mapping Laboratory, University of Georgia, Rm. 162, Riverbend Research Center, 110 Riverbend Rd., Athens, GA 30602
^d Department of Plant Pathology and Microbiology, Texas A&M University Agricultural Experiment Station, 2415 East Highway 83, Weslaco, TX 78596

View larger version (18K): [in a new window]   Fig. 1. Schematic representation of the GUS fusion plasmids linearized at the unique HindIII site showing the different promoters, locations of exons and introns, and the nos termination sequence (nos) cloned into pUC8 or pUC19. Restriction sites: E, EcoRI; H, HindIII. Numerical values indicate approximate kilobase pairs.

View larger version (143K): [in a new window]   Fig. 2. Genomic library screening with first strand cDNA. The arrow indicates a genomic clone to be characterized further.

View larger version (67K): [in a new window]   Fig. 3. Restriction enzyme digestions of phage DNA from 12 genomic clones (A). Each clone was digested with BamHI, EcoRI, or BamHI + EcoRI in duplicate lanes for each enzyme digestion. Lane 1, 40, 41 and 80: HindIII; lane 38 and 60: 1-kb ladder. Lane 2–7: clone 8-1; lane 8–13: clone 9-1; lane 14–19: clone 9-2; lane 20–25: clone 10-1; lane 26–31: clone 14-1; lane 32–37: clone 14-2; lane 42–47: 15-1; lane 48–53: 16-1; lane 54–59: 17-2; lane 61–66: 18-1; lane 67–72: 19-1; lane73–75: clone 21-1. The Southern blot was hybridized with pooled first strand cDNA derived from mRNA, the signals (B) from five identified genes including sugarcane tubulin (STUB), sugarcane ubiquitin (SUbi), sugarcane elongation factor (SEF1), sugarcane aquaporin (SAQ), sugarcane proline-rich protein (SPRP1) are marked by arrows.

View larger version (68K): [in a new window]   Fig. 4. Northern analysis of four cDNA clones isolated by genomic and cDNA library screening. Total RNA was isolated from young roots (R), stems (S), and leaves (L). The 18S rRNA signal was used for standardizing the signal level per lane. Autoradiographs were exposed for 1 d for STUB, half day for SPRP1 and 3 d for SAQ1 and SEF1.

View larger version (31K): [in a new window]   Fig. 5. Comparison of promoter strength in sugarcane embryogenic callus. Flourometric GUS assays were replicated three times for each treatment and each treatment was replicated three times. Vertical bars indicate the SE of the nine data points used to calculate each of the treatment means.

View larger version (133K): [in a new window]   Fig. 6. Embryogenic wheat callus showing GUS activity after bombardment with promoter-GUS fusion constructs. (A) Promoter SPRP1, (B) Promoter SPRP 2.4, (C) Promoter SEF1, and (D) maize ubiquitin-1.