RESULTS
The proteins were first isolated. Since different types of plant tissue vary in the amount of protein contained, we used a standard curve to determine the concentrations of each protein sample (Figure 1). The absorbance at 760 nm was recorded for various dilutions of each protein sample. The protein dilution with an absorbance reading between 0.125 and 1.25 was used for finding the concentration. Hibiscus and gooseberry tissue both had relatively high concentrations, so the absorbance of a 1:20 dilution was used to find the concentrations. A 1:2 dilution was used for mother-in-law’s tongue and papyrus to obtain the concentrations (Table 1).
| Tissue | Concentration (ug/mL) |
| Hibiscus | 1.5 |
| Papyrus |
2.63 |
| Mother-in-Law's Tongue | 1.6 |
| Gooseberry | 0.55 |
In order to make sure there were proteins in our sample and that they did not get degraded during the isolation process. All protein samples were run on an SDS-PAGE gel (Figure 2). Each contained protein with papyrus having the least amount. The large subunit of Rubisco has a molecular weight of 55 kD and was used to represent the rubisco protein complex. According to the gel, the mother-in-law’s tongue, and hibiscus had visible rubisco proteins.
A western blot of an SDS-PAGE gel of each tissue’s protein samples was done in order to see if rubisco was present (Figure 3). A small amount of rubisco protein appears to be found in the mother-in-law’s sample. A significant amount of rubisco was found in the papyrus and hibiscus protein samples and only an extremely slight amount appears to be found in the gooseberry protein sample.
In order to determine the mass of DNA found in each sample, a Real Time PCR was run. Standards of known concentrations were run to give a logarithmic curve of the CT values (Figure 4). Figure 5 displays the results from the Real Time PCR. The point at which the fluorescence rose to the threshold line was the CT value of the sample. This means that the PCR is recording DNA instead of background. The threshold line was determined by lining it just above the control sample with no DNA. The equation in Figure 4 was used to find the concentration of the four samples by plugging in each CT value in for the y value (Table 2). CT values were given by the Opticon Monitor program which displayed the Real Time PCR results. It was found that the gooseberry had the highest amount of the rubisco gene; while papyrus had the lowest amount. The hibiscus and mother-in-law's tongue had similar amounts in between gooseberry and papyrus. Graph B in Figure 5 displays that all of the samples have approximately the same melting points at 83oC compared to the no DNA control at about 65oC. It is safe to conclude that the double stranded DNA hybrids formed in the PCR were the same in each sample.
| Plant |
Mass (ug) |
| Hibiscus | 0.00154 |
| Gooseberry | 0.0767 |
| Papyrus | 0.00001489 |
| Mother-in-Law's Tongue |
0.000464  |
In order to be sure that the DNA hybrids formed in each sample was amplified from the rubisco gene, an agarose gel was run. The fragment size of the set of primers should have produced a double strand 200 base pairs long. The gel in Figure 6 shows that all four samples of amplified double stranded DNA of interest were 200 base pairs (bp) long.
