Discussion

Western Blot

In isolating the protein for the Western Blot procedure, there were usable amounts of protein as shown by the spectrophotometer data in Table 1. However, the membrane from the Western Blot, as well the gel from the Comassie Blue stain both showed no protein (Figure 2). Since the Comassie gel showed no protein, it can be inferred that no protein was in the Western Blot either, and so the antibodies had no RBCL with which to bind. Therefore, no conclusions can be drawn from this data.

There may have been several reasons for the outcome of this data. One possible reason is that the concentrations determined for the protein may have been wrong. The standard curve produced for the protein may have been slightly skewed, since one of the points (1.25mg/mL) had to be removed and the R² value was still only 0.9299 and not in the desired range of >0.98. Other than this, the protein may have been degraded by proteases while in storage for the five week period. As far as the actual Western Blot procedure, there is a possibility that errors may have occurred along the way in the preparation of the protein. This could be a significant factor, since the protein standard RBCL did not show any results on either the stained gel of the Western Blot membrane.

The last possibility as to what may have occurred could be that there was in fact protein, but since the Comassie stained gel was a bit blurry, the protein may not have shown. If this was the case, it would also mean that there was no RBCL in the protein, since no antibodies were detected on the membrane. However, going back to the fact that the protein standard RBCL did not show up on the membrane either, this is most likely not the case.

DNA Isolation

Usable quantities of DNA were able to be isolated from the four samples (Table 3).  The purity of this DNA is also of importance.  Good quality DNA without significant amounts of protein has an A₂₆₀/A₂₈₀ ratio of 1.8-2.0.  All of the samples except the (flowers,water) sample had an A₂₆₀/A₂₈₀ ratio within this range. The ratio for this sample was 1.00. An A₂₆₀/A₂₈₀ ratio of 1.00 is indicative of lots of protein contamination in the DNA.  Thus it could be concluded that the low purity of the 1:28 (flowers, water) dilution is due to large amounts of protein still present in the sample.

Due to the DNA concentration for the (flowers,water) 1:28 dilution being very low (3.2 ng/µL), with an A₂₆₀/A₂₈₀ ratio of 1.00, this DNA sample was tested again at a dilution factor of 1:2.  After the new dilution (1:2) was performed, the DNA concentration increased to 22.2 µg/µL, but the purity increased to the abnormally large number 23.84. This number could have been due to random error in the sample, or in the instrumentation used.

Based upon the results from real-time PCR, it cannot successfully be concluded which of the four samples contained the most RBCL DNA.  The log scale graph of our plate read indicates that the negative control (no DNA sample) had a C_t value of approximately 21 (Figure 3).  The negative control should not have crossed the threshold line.  The most likely cause of this is that there was some DNA contamination in the negative control thus possible DNA contamination between the unknown samples as well.  The agarose gel electrophoresis results further lend support to the possibility of DNA contamination (Figure 5).  The negative control (lane 5) exhibited several distinct PCR products which could only be explained by DNA contamination of the negative control.

The melting curve of the negative control in Figure 4 is very similar to the melting curves of the unknown samples.  The negative control had a melting temperature of 83ºC while all unknown samples had a melting temperature of 84ºC (Figure 4).  DNA contamination is most likely responsible for the results obtained.  Ideally, the negative control should only contain primer dimers and thus melt off at lower temperature than the actual DNA samples.  However, the conclusion can be made that the same type of DNA was amplified in all of the unknown samples because the melting temperature was almost identical for all of these samples (Figure 4).

Future Research

Future research would include performing the experiments again with the modification of performing the Western Blot within 24 hours of isolating the Rubisco protein; storing the isolated Rubisco protein for a much shorter time period would reduce the probability of proteases degrading the protein.  Future research should also include investigating the effects of varying the time of desiccation on the expression of RBCL; one could look at how RBCL expression varies in chrysanthemums that have been exposed to 2, 4, 8, and 16 days of drought (no watering).

Conclusion

Based upon the data collected, no definitive conclusions could be made about where RBCL is expressed in chrysanthemums (leaves versus flowers) or how the amount of moisture present affects the amount of RBCL expressed in chrysanthemums (leaves versus flowers).