Isolation of the polyubiquitin promoter from Gladiolus GUBQ1, (GenBank accession DQ445914) using the Genome Walker Kit (Clontech) was previously described . The 1.9 kb GUBQ1 promoter (G1-1) and a 0.68 kb intronless version of this promoter (G1-3) were previously subcloned into pUC-GUS and used for transient GUS expression in callus . For Arabidopsis transformations, the GUBQ1 promoter and its intronless version were subcloned into the binary vector pCAMBIA1391Z. For the transient expression assay of tobacco, either G1-1(A) or G1-3 were subcloned into the binary vector pBI121-GFP which contained the gfp gene instead of the uidA gene (Figure 1). G1-1 contains 1898 bp which consists of 600 bp promoter (Gubi1-P), 80 bp 5′UTR-exon (5′U-E), and 1218 bp 5′UTR-intron (5′U-I). In G1-1 (A), the 1218 bp 5′U-I of GUBQ1 has been substituted with the Arabidopsis actin7 gene 5′UTR-intron (541 bp).
The 5′UTR was identified using the 5′RACE & 3′RACE kit (Clontech). Total RNA was isolated from Gladiolus and 2 μg used for RT PCR according to the manufacturer’s directions. AP1, an adaptor-specific primer present in the kit, and a ubiquitin gene-specific primer (RT: G1-1 and AS: AGG AAT CCC CTC CTT GTC CTG G) were used for the first PCR reaction of 5′ RACE. The second PCR reaction used AP2, an adaptor-specific primer in the kit, and a ubiquitin gene-specific primer (RT:G1-2 and AS: ATC GAT TGT GTC GGA GCT CTC C). PCR products were cloned into a pTOPO-TA vector (Invitrogen) for sequencing. The 5′UTR sequence was revealed by sequencing and then verified by showing that this sequence was spliced out.
Suspension cells of Gladiolus cv. Jenny Lee were transformed using the PDS-1000/He gene gun (Bio-Rad, Hercules, CA, http://www.bio-rad.com) . Callus was initiated from plants growing in vitro on Murashige and Skoog’s medium (MS)  containing 3% sucrose, 1.0 mg/l glycine, 100 mg/l m-inositol, 1.0 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 0.5 mg/l (2.2 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2% Phytagel (Sigma-Aldrich, http://www.sigmaaldrich.com). The callus was used to start suspension cells that were grown in the same medium lacking Phytagel. Both callus and suspension cells were grown in the dark at 25°C. Suspension cells were grown on a gyratory shaker at 120 rpm and transferred every 2 weeks by placing half of the cells into 30 ml of fresh medium.
Suspension cells were co-bombarded at 1200 psi (8.3 MPa) with p35SAc that contains the selectable marker gene phosphinothricin acetyltransferase (PAT) under control of the CaMV 35 S promoter (received from AgrEvo, Somerville, NY) and one of the promoter plasmid DNAs (CaMV 35 S, G1-1, or G1-3). One week after bombardment, cells were placed on MS medium as described for callus initiation supplemented with 3 mg/l bialaphos (Meiji Co., http://www.meiji.co.jp) for selection. Callus was transferred monthly to fresh selection medium, and after about 4 months, surviving callus pieces were transferred to regeneration medium. Regeneration medium is the same as callus initiation medium except with 2 mg/l (9.3 μM) kinetin rather than 2,4-D, and supplemented with 1 mg/l bialaphos. Plantlets recovered after growth on regeneration medium were grown on MS medium without hormones and without bialaphos. The cultures were grown under a 12-h photoperiod using cool-white fluorescent lights (40–60 μmol·m2·s1).
Transformation of Arabidopsis thaliana ecotype Columbia plants grown 5 weeks in a growth chamber with a 16 h photoperiod at 22°C were transformed with Agrobacterium tumefaciens strain C58C1 using vacuum infiltration . Hygromycin-resistant T1 plants were selected by planting seeds on MS medium supplemented with 30 mg/l hygromycin. Hygromycin resistant seedlings were then transferred to soil and transgenic plants were verified to be transformed by PCR. The gus gene was amplified using the primer sequences 5′-GTT GGG CAG GCC AGC GTA TCG TG-3′ and 5′-TAA CCT TCA CCC GGT TGC CAG AGG-3′ and thermal cycler conditions of: 1 cycle of 95°C for 5 min and 35 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 1 min, followed by 3 min at 72°C.
Agrobacterium tumefaciens strain GV3101 was used for transient expression of tobacco (Nicotiana benthamiana). Agrobacterium was cultured for 16 h in liquid medium. Cells were then centrifuged and resuspended in 10 mM MgCl2 at an OD600 of 0.9. Resuspended Agrobacterium cells were vacuum infiltrated into tobacco leaves that had been collected from tobacco plants grown in soil at 22 ~ 24°C for 4-5 weeks under a 16 h light photoperiod. Tobacco leaves were harvested 3 days after vacuum infiltration and used for gfp expression analysis.
RNA isolation and RT-PCR
RNA was isolated from plant tissues using the Qiagen RNeasy kit (Qiagen Inc, Valencia, CA). The isolated RNA was treated with RNase-free DNase (Roche) to remove DNA. Reverse transcription was performed using 1 μg total RNA and 20 μl reaction mix of the iNtRON Power cDNA synthesis Kit (Intronbiotech, http://www.intronbio.com) and AMV reverse transcriptase (Intronbiotech) according to the manufacturer’s directions. For identification of 5′UTR-intron splicing in transgenic plants, RT-PCR was performed with the 5′UTR exon specific primer, 5′U-E F (5′-AGG GTT TTC TCA TCC CCA AAT T-3′) and either a uidA or gfp gene specific primer, uidA 200 R (5′- CCT GAT GCT CCA TCA CTT CCT G-3′)/gfp R (5′-AGA AGA TGG TGC GCT CCT GG-3′). For the RT-PCR, 2 μl of each primer (0.2 mM final concentration), and 2 μl of the cDNA preparation were used in the reaction mix (25 μl final volume). The thermal cycler conditions of: 1 cycle of 95°C for 5 min and 30 cycles of 94°C for 20 s, 55°C for 20 sec, 72°C for 1 min. RT-PCR products were cloned into a pTOPO-TA vector (Invitrogen) for sequencing. Quantitative PCR amplification was performed with gene-specific primers and the SYBR Green Master Mix kit (Qiagen) using a Rotor-Gene 6000 real time amplification operator (Corbett Research, Mortlake, Australia). The reaction mix (25 μl final volume) consisted of 12.5 μl of the SYBR Green PCR Master Mix, 2 μl of each primer (0.2 mM final concentration), and 2 μl of the cDNA preparation. The thermal cycler conditions of: 1 cycle of 95°C for 15 min and 45 cycles of 94°C for 20 s, 55°C for 20 sec, 72°C for 30s.
In real-time PCR, the actin gene specific to each plants species being analyzed was used as the reference gene and to determine the possibility of DNA contamination. The actin primer sequences included the following: 1) for the Gladiolus actin gene 5′-CTG CCA TGT ATG TTG CAA TCC A-3′ and 5′-GGA AGA GGA CTT CAG GGC ACC TG-3′ (384 bp), 2) for the Arabidopsis actin gene 5′-GTC CCT GCC ATG TAT GTT GCC-3′, and 5′-GTG GTG AAC ATG TAA CCT CTC-3′ (200 bp), and 3) for the Nicotiana actin gene 5′-TTG GAA TGG AAG CTG CTG GA-3′, and 5′-TCA GGA GGC GCC ACC ACC TT-3′ (202 bp).
All Samples were run three times in triplicate.
Arabidopsis Actin7 gene 5’-UTR intron isolation
The 5′-UTR intron of actin7 gene (Genebank ID, U27811) was isolated from genomic DNA of Arabidopsis thaliana ecotype Columbia. PCR amplification of the 5′-UTR intron was accomplished using the primer sequences 5′-AGG TGA GTC TCT AGA TCC G-3′ and 5′-CCT AAA AAA AAA GTA AAA TGA AAC-3′ and thermal cycler conditions of: 1 cycle of 95°C for 5 min and 35 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, followed by 3 min at 72°C. PCR products were cloned and sequenced.
The specific activity of GUS expression was determined by fluorometric determination of methylumbelliferone (4-MU) . The extraction buffer consisted of 100 mM KH2PO4, pH 7.0 and 1 mM dithiothreitol. Extracts were centrifuged at 16,000 × g at 4°C for 12 min, and a 50 μl aliquot was added to 0.5 ml of assay buffer (1 mM methylumbelliferyl-β-D-glucuronide) for incubation at 37°C. A 100 μl aliquot of the assay buffer containing cell extract was added to 0.9 ml of 0.2 M sodium carbonate after incubation for different lengths of time. Fluorescence of 4-MU was measured using a Bio-Rad VersaFluor Fluorimeter set at 360/40 nm excitation and 460/10 nm emission. The amount of protein in the cell extract was determined using the bicinchoninic protein assay reagent (Pierce, http://www.piercenet.com).
Transgenic plants were assayed for expression of the gus gene that codes for β-glucuronidase (GUS) following the histochemical staining procedure described by Jefferson et al.  with some modifications. Shoots were incubated 16 h at 37°C in staining solution [100 mM sodium phosphate, pH 7, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM Na2EDTA, 0.5% (v/v) Triton X-100] with 0.5 mg/l 5-bromo-4-chloro-3-indoly-β-D-glucuronide. After 16 h of staining, the shoots were cleared and fixed in a solution of 75% (v/v) ethanol:1% (v/v) acetic acid.
Three independently transformed plant lines of Gladiolus growing in vitro or three lines of Arabidopsis were analyzed for each DNA construct, and three plants were analyzed for each transgenic line. Three plants of each plant line were used for the mean determination of GUS expression and standard error calculated.
For real time PCR the relative amount of target RNA for each sample was calculated by the statistical analysis method .
Genomic DNA for Southern hybridizations was isolated from plants using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was digested with Hind III, electrophoresed on a 0.8% agarose gel in 0.5× TBE buffer, and then transferred to a nitrocellulose membrane. For hybridization a 492 bp PCR-amplified gusA gene product was labeled using the PCR DIG Probe Synthesis kit (Roche, http://www.roche.com) and used as the probe. Hybridization and detection of the DIG labeled nucleic acid was performed using the DIG Easy Hyb and DIG Nucleic Acid Detection kits (Roche).
Plant leaves were ground in Bradley buffer (50 mM Tris–HCl pH 7.5, 10 mM KCl, 20% glycerol, 0.4 M sucrose, 5 mM MgCl2, 10 mM β–mercaptoethanol, 1 mM PMSF) and centrifuged for 20 min at 13,000 × g, 4°C. The supernatant was used for SDS-PAGE gel electrophoresis, and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with blocking solution (Tris-buffered saline solution pH 7.4, 0.1% Tween 20, 5% nonfat milk). The GFP detection rabbit anti-GFP polyclonal antibody was used as the primary antibody, and anti-rabbit horseradish peroxidase-conjugated was used as the secondary antibody. Immunobiochemical detection was performed using the ECL Plus detection system (Millipore). For quantification of GFP the Multi Gauge Version 3.0 program was used following exposure of the Western blot to Fuji film.