Table 5. Specific Ct values for each 50 ng tissue sample. Ct values indicate the point at which the fluorescence passes the threshold.
Results
Figure 5. 2% agarose gel electrophoresis of 50 ng petal and leaf DNA samples from white, orange and purple chrysanthemum. Gel stained with ethidium bromide and imaged with UV light. Lane assignments: 1 Kb plus ladder (ladder), control (C), purple petal (PP), purple leaf (PL), orange petal (OP), orange leaf (OL), white petal (WP), and white leaf (WL).
Figure 4. Real-Time PCR data from 25 ng petal and leaf DNA samples from white, orange and purple chrysanthemum. Increased fluorescence represents increased DNA content. Cycle indicates PCR cycle number.
Figure 3. Melting temperature data during real-time PCR for the 25 ng samples of petal and leaf tissue from white, orange and purple chrysanthemum. All tissue samples peak around 83 °C. The yellow, double-peaked line indicates the blank.
To verify that the amplified DNA was the intended 200 bp fragment, agarose gel electrophoresis was performed on the amplified products. Because PCR of the 25 ng samples amplified all tissues, this concentration was selected for gel electrophoresis. Errors in running buffer concentration corrupted the procedure, so it was repeated with the 50 ng samples. Imaging reveals a 200-bp band for the three leaf samples and the control sample, with the purple leaf the strongest, followed by the white leaf, then the orange (Figure 5). The gel results corroborates the Real-Time PCR data, only the DNA from the leaf samples was amplified (Table 5).
Table 4. Specific Ct values for each 25 ng tissue sample (including blank). Ct values indicate the point at which the fluorescence passes the threshold.
Table 2. DNA concentration and purity for petal and leaf samples of white, orange and purple chrysanthemum. Absorbance measured at 260 nm and 280 nm. DNA purity measured by A260/A280 ratio.
Figure 1. Protein standard concentration curve. Each point represents a specific protein standard's absorbance at 750 nm. The slope formula generated by the curve was used to calculate the protein concentrations in the petal and leaf tissue samples presented in Table 1.
Real-time PCR was used to quantify RUBISCO DNA in the tissue samples. Melting curve graphs were generated to establish the temperature at which to examine the fluorescent signals during each cycle. For the 25 ng samples, the signals peaked at about 83°C, whereas the control demonstrated a double peak at 80°C and 86°C (Figure 3). By selecting a temperature of 81°C, the formation of primer-dimers were effectively minimized in the Ct analysis. For the 50 ng samples, 82°C was used. Ct values, or the PCR cycle when the signal is amplified beyond the background, can be used to compare relative initial concentrations of the amplified gene. Simultaneous amplification of standards with a known concentration can provide specific initial concentrations of the amplified gene in the samples. Amplification of the 25 ng samples revealed Ct values for all tissue samples, including the blank (Figure 4). Similar Ct values from the leaf samples (11.0-12.6) were observed, indicating similar initial DNA concentrations. Petal samples produced similar Ct values (14.3-15.8) that were somewhat higher than the leaf samples, indicating somewhat lower initial DNA concentrations (Table 4). Amplification of the 50 ng samples revealed Ct values for the leaf samples only, which were wide ranging (Table 5). Initial DNA concentrations could not be determined, because the standard samples used during PCR produced aberrant results (data not shown).
Purple Petal
White petal
Orange Petal
White Leaf
Orange Leaf
Purple Leaf
Blank
RUBISCO Protein analysis
To determine protein concentration in the petal and leaf tissue of white, orange and purple chrysanthemums, diluted (1:2, 1:20, and 1:200) protein samples were assayed, then absorbance readings were taken at 750 nm. Standard protein samples with known concentrations were similarly assayed. For the standard protein samples, an absorbance/concentration curve was generated (Figure 1). The coefficient of determination (R2) indicated a high degree (.9258) of correlation between absorbance and protein concentration. The absorbance readings of the 1:200 dilutions from the petal and leaf samples fell within the standards range, so those values were used to calculate the protein concentration in each undiluted sample (Table 1).
To compare the RUBISCO protein content among our tissue samples, 30 ug protein aliquots from each tissue were loaded into two 10% polyacrylamide gels along with 3 mL of RUBISCO standard and a molecular weight marker. Imaging from the first gel stained with Comassie Blue did not reveal protein from any of the samples, including the standard (image not shown). Imaging from the Western blot revealed three poorly visualized antibody bands from all three leaf samples, but nothing from the petal samples. These could not be confirmed as RUBISCO proteins, because the standard sample did not reveal antibody luminescence for the large subunit (Figure 2).
Figure 2. Western blot for RUBISCO large subunit in petal and leaf tissue samples from white, orange and purple chrysanthemum. Nitrocellulose membrane was washed in RUBISCO large subunit chicken antibody, then anti-chicken antibody tagged with horse radish peroxidase, then exposed to X-ray film for 1 hour and photographed with chemiluminescent camera. Lane abbreviations: PP (purple petal), PL (purple leaf), OP (orange petal), OL (orange leaf), WP (white petal), WL (white leaf). Blue carets indicate position of reactive proteins.
Table 1. Leaf and petal tissue protein concentrations from white, orange and purple chrysanthemum. Absorbance readings at 750 nm of diluted (1:200) protein samples were inserted into the standard curve equation (concentration = 4.9128 x absorbance) to get diluted concentration, then multiplied by the dilution factor (200) to get stock concentration.
To obtain DNA concentrations in each petal and leaf tissue sample and to determine its purity, absorbance readings were taken for each sample at 260 nm (nucleic acid) and 280 nm (protein). The ratio of nucleic acid to protein (260/280) indicated the purity while absorbance was used to calculate concentration were as described in the methods section. All of the petal and leaf samples produced ratios above the standard 1.8-2.0 range for a relatively pure DNA sample and the DNA concentrations were uniformly low (53.7-119.4 ng/mL) (Table 2).
| Tissue Sample | Ct Value |
| Purple petal | None |
| White Petal | None |
| Orange petal | None |
| White Leaf | 12.34 |
| Orange Leaf | 29.21 |
| Purple Leaf | 11.14 |
