R u b i s c o
DISCUSSION
The purpose of this study is to determine if the amount of Rubisco decreases with age. To examine this, we conducted a quantitative PCR (qPCR) analysis on chloroplastic DNA that codes for the large subunit, an sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel, and a Western Blot analysis. We hypothesized that the Rubisco large sub-unit levels would be higher the younger the plant tissue was.
Although it was not noted in our results section, all DNA extracts received a A260/A280 purity between 1.8-2.0 showing that our DNA was of good quality and could therefore good produce dependable results in qPCR.
The results in Table 2 show that for the rbcl2f primer set, the ranking from highest to lowest Rubisco DNA content is six, four, ten and eight week tissues. Primer set rbcl2F followed the same trend as well with the order from highest to lowest levels of Rubisco DNA. After observing the qPCR graphs (Figures 3 and 4) a trend can be seen that the Rubisco induction is in fact higher in the four and six week plants than the eight and ten week plants. This data is displayed in Figure 7 and makes data analysis much easier. Keep in mind that the lower the Ct value the higher the relative content of DNA. However, this data does not match our hypothesized trend sample to sample. We did not see an overall decrease, sample to sample in Rubisco large-subunit levels, just a trend that the first two samples, weeks four and six, had higher Rubisco large-subunit levels than the last two samples, weeks eight and ten. It is important to keep in mind that these levels do not reflect Rubisco levels directly, but indirectly. Regulation of transcription and translation can greatly effect the final Rubisco levels. The data presented by the qPCR allows the prediction of final Rubisco levels, and this is all that can be used by our lab group seeing that the western blot was unsuccessful. The controls for the qPCR experiment showed very little DNA presence and the only presence seen was probably due to primer dimers which presence is foretold by the early peak seen in Figures 5 and 6 (for primer set C at about 80ºC and primer set A at about 75ºC). This was accounted for by taking the readings for the plate at 81ºC which was above the primer dimer melting point but below the DNA melting points for both primer sets.
The PCR products where run on an agarose gel (Figure 8) and the segments for each primer match up well. The DNA segments for both primers ran at the expected size for the primer used. This gel was run to show that our PCR product was in fact the correct size, but since there is product showing up in the "no template controls" (NTC) contamination seems to be an issue. This issue may be due to well spill over, but more likely is due to DNA contamination of the NTC samples. This contamination can also be seen on our melting curves in the NTC, but this contamination may have been amplified in these NTC sample because there was no real template to amplify. Note that these contamination peaks are only seen in the NTC. This could have had altering affects on our data but the best we can do is assume that the contamination did not effect qPCR too greatly. In lane 1 primer dimers can be seen but these cause no imminent problems.
Our SDS-PAGE gel (Figure 9) is very difficult to interpret. We would assume that the protein on the gel was Rubisco, but since the ladder does not contain all five bands, and the fact that the photo is in black and white (the markers are color coded), we cannot tell what size this isolated protein is. It does appear to be near the top band which would make it well over 150 kDa and Rubisco is ~55 kDa. So this protein very well could not be Rubisco after all. The peculiar appearance of our bands may be due to a bizarre contaminant in the protein sample or due to overloading the gel. This conclusion is based off of the fact that the ladder does not contain the same "V" shape that the bands do. The western blot resulted in a blank gel; even the molecular weight did not transfer. The buffer solution used during the transfer to the membrane was TTBS and not transfer buffer as we should have. Having no proteins on the membrane gel resulted in no antibodies binding and therefore a blank membrane.
The hypothesis that Rubisco levels would decrease with age is partially consistent with the qPCR data. The week four and week six plants do have significantly higher Rubisco large-subunit levels than weeks eight and ten when looking at the qPCR data. This clear difference between the first two samples and the last two samples may be hinting towards a difference in Rubisco large-subunit regulation as the plant ages or enters senescence. This is the only data available to verify our hypothesis. Without a western blot this is the only data that can be used to verify the hypothesis. This is unfortunate because so many levels of regulation including degradation, transcriptional, and translational affect the final protein levels in vivo. This conclusion is pending that the protein isolated in the first place was Rubisco large-subunit and not some other protein which the SDS-PAGE gel may hint that it is.
If we indeed had a Western Blot to analyze, would the levels be different than those seen in the qPCR? This question is hard to answer. If Rubisco is largely regulated via translation and degradation these levels could be entirely different. That is why looking at DNA is hard to draw convincing conclusions on. Eukaryotes in general have a huge amount of fine-tuning in their regulation of expression on the track from DNA to protein (and even once the protein is made), so using DNA to compare relative amounts of a protein in tissues may not be very insightful at all.
Further Research:
If this experiment were to be repeated, our lab group would like to attempt at achieving better results on the SDS- PAGE gel to verify our results. We would also like another shot at using western blot because our initial attempt failed. Running qPCR again may prove whether our results are repeatable which may help verify that they are real. Another qPCR may be ran using isolated cDNA to check and see how this compares to the genomic DNA qPCR data. Cholorplastic DNA is not an indication of the amount of Rubisco protein present because Rubisco is a highly regulated protein on all levels: transcription, translation and even degradation (the most prominent form of control during senescence) (Suzuki et al. 2001).