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The two species of plants used in the study of Rubisco expression, the common Ice Plant, Carpobrotus edulis (formerly classified as Mesembryanthemum crystallinum), and Oak tree leaves, Quercus alba, were chosen because Ice Plants are known to lower the pH of soil which should lower Rubisco levels. Rubisco has a high optimal pH; therefore, it was hypothesized that Oak leaves would have higher Rubisco expression than the Ice Plant. The difference between Rubisco expression was also compared between samples taken during the night (6:00 am) and the day (12:00 pm). Rubisco is mainly used during the day in C3 plants; therefore, in addition to differences between plant species, we also hypothesized that Rubisco levels would be higher during the day than during the night. The conditions under which our samples were gathered, however, were not ideal because they were collected right after a long period with very little rain and it was cloudy when the samples were collected during the day. The cloudy conditions may have reduced the amount of Rubsico expression as compared to the levels on a bright sunny day. Ice plants also have the ability to switch from C3 to CAM, in which they carbon fixate primarily at night, in dry conditions. Though the pH of the soil was not measured, the pH can somewhat be taken into account with lowering Rubisco levels, though the hypothesis cannot be based solely on this observation. After the DNA was separated and purified, and the total DNA concentration and purity levels were determined (Table 5 and 6) the Real Time PCR reactions were able to be set up. The two different primer sets, rbc12f/RBCL-fonfana and rbcl2F/RBCL-Savolainen, were selected because: there was no indication that they would not work on our plant species, they produce smaller products, and just in case one primer did not work well. We chose to run 100ng of each of our DNA samples because that was the median of the recommended amount to use, and we chose to run an additional reaction of the DO and NO samples with only 50ng of DNA because of the low purity levels of these samples shown in Table 6. Because of the low purity levels it was expected that these samples would have less DNA. It was expected that these samples had low purity levels due to difficulty in grinding. The PCR data produced from the first primer set, rbc12f/RBCL-fonfana, shown in Table 7 and Figures 3 and 4, produced better results than the data produced from the second primer set, rbcl2F/RBCL-Savolainen, shown in Table 8 and Figures 5 and 6. This can be determined by comparing the melting curves shown in Figures 4 and 6. The melting curve in Figure 4 of the reactions run with the primer set rbc12f/RBCL-fonfana, showed the blank sample likely containing primer dimers melting off around 76º Celsius, and then the other samples melting off together around 82º Celsius. This indicates that if the reactions are read at 82º Celsius to get the graph shown in Figure 3, then the samples are amplifying correctly producing the desired products. In other words, only our samples are being read with primer dimers, and anything in the blank sample not interfering with the data. The melting curve in Figure 6 does not show as good of results with four of the samples melting off with the blank sample. This data would make it difficult to determine what temperature to take the data at shown in Figure 5 because it is not clear if the correct samples were being amplified producing the desired products. The undesired trend observed in this melting curve could have been because the run got stopped early on accident. The data produced from the agarose gel electrophoresis shown in Figure 7, however, indicates that both primers ran correctly because the first three lanes containing samples from primer set rbcl2F/RBCL-Savolainen produced fragments around 200bp and the last three lanes containing samples from primer set rbc12f/RBCL-fonfana produced fragments around 500bp. These were the predicted band sizes that should have been produced; however, we focused on the data set produced by the primer set rbc12f/RBCL-fonfana because of the melting curve. The data from Table 7 and Figure 3 resulted in the following rank from greatest to least chloroplast DNA that should contain Rubisco large subunit (from the 100ng samples): NI, DO, NO, DI. The DO and NO samples with only 50ng of DNA resulted as expected with a similar trend as the DO and NO samples with 100ng of DNA, but having a lower chloroplast DNA expression since they contained less DNA. It would have been most beneficial to also run 50 ng of NI and DI samples to see if purity really was a problem, but there were space constraints in number of samples that could be run in PCR. Therefore, the 100ng samples will be emphasized. The results of the PCR support our hypothesis that DO would have higher Rubisco expression than NO with Ct values of 10.133 and 13.346 respectively; however, our hypothesis that the oak leaves would have greater Rubisco expression than the Ice Plant, and that DI would have greater expression than NI, was not supported. We hypothesized that since Rubisco levels are highest when a plant is carbon fixating, that Rubisco expression would be the highest during the day because that is when C3 plants do their carbon fixation. This is supported by the results from the Oak plant. An explanation for why the ice plant had higher expression during the night than during the day could be that it had switched to a CAM plant which performs carbon fixation during the night. As mentioned previously, Ice Plants are known to switch from C3 to CAM in dry conditions, which could have resulted from the long period with no rain. An explanation for why the oak leaves didn’t have greater Rubisco expression than the ice plant is that when the DNA was extracted, we had some difficulty separating the DNA from the proteins in the oak samples. This is supported by the purity levels shown in Table 6 with the DO and NO samples having a purity level of 1.56 and 1.24, which is significantly below the desired level of 1.8-2. The proteins in our DNA samples could have interfered with the PCR reactions affecting the level of expression. With the melting curve in Figure 6 showing that our samples might not have amplified correctly, the data in Table 8 and Figure 5 don’t give very useful results. From the melting curve of this data the only samples that appear to be amplifying correctly are the DI and NI samples. They produced products showing that NI had greater chloroplast expression than DI. This is the same trend that the data set from the first primer produced when comparing NI to DI, supporting again that the ice plant may have switched to a CAM plant. From these results, since the ice plant appeared to have switched from a C3 plant to a CAM plant, it is difficult to make any conclusions about the effect of pH on Rubisco expression. With the PCR results previously mentioned, the level of Rubisco large subunit expression could be determined indirectly by quantifying the amount of chloroplast DNA. This could be done because the gene for Rubisco large subunit is expressed on chloroplast DNA. In doing so it doesn’t take into account the fact that there could be different Rubisco levels in the chloroplasts between samples. Therefore, in order to further quantify Rubisco levels and determine that we did have the Rubisco protein, protein analysis was performed. This was done to see if there were any further changes in expression that were contributing to protein levels. Protein from each sample was loaded onto a gel and then a Coomassie blue stain was used on the gel to detect protein to determine if a band the size of Rubisco large subunit formed, and also how much protein was present. This gave inconclusive results shown in the picture in Figure 2. This could have resulted because we were only able to load a very small amount of protein as a result of our total protein concentrations being low and also because a Coomassie blue stain is not very sensitive. The Western Blot that used antibody detection to detect protein product did not produce any results either with no product or marker being visible on our membrane. This procedure was supposed to be 1000-fold more sensitive than the Coomassie blue stain; therefore, it was expected that even though only a small amount of protein was able to be run on the gel, it would have still produced products. Most likely the proteins did not transfer from the gel onto the membrane. This can be assumed because if everything was transferred correctly, the marker would have at least faintly shown up even if there was no protein product. The results from the protein analysis do not support or negate our hypothesis. Overall, the only results that were useful were obtained from PCR. The Coomassie blue gel and the Western Blot did not produce any results. A possibility that no proteins appeared on the Coomassie blue gel was because the stain is not as sensitive as antibodies. A very small amount of protein was used in the two gels, and though it was thought that bands would appear on the Western Blot, they also did not. The Western Blot was to be 1000 times more sensitive than the Coomassie blue due to the use of antibodies. A reason that there were no bands on the Western Blot membrane was due to the fact that the membrane was laid down part way onto the gel, and then picked up, and laid again. The proteins could have immediately transferred from the gel onto the membrane, and thus would not show up when the membrane was photographed. Another point to add to the Western Blot procedure is the membrane was not translucent when it was removed from the methanol, as it should have been. This could have also prevented the protein transfer. In addition, the purity of the DNA, as previously stated, was not very good for the Oak samples. This experiment could be repeated using the same plants, but being extra careful with such errors as previously mentioned, and also pipetting errors and correcting any inhibitions there could be. Hopefully, better results would be obtained. To further support the hypothesis, another possibility would be to try a different plant, rather than Oak, having a fleshier leaf, or even stem. The Oak was very difficult to grind up, as opposed to the Ice Plant. Overall, despite our lack of conclusive results in the Western Blot, the PCR did support our hypothesis that the Oak leaves would have more Rubisco produced during the day than at night. Works Cited 1. Kore-eda,
Shin and Cushman, Mary Ann et al. Transcript profiling of salinity
stress 2. DeRocher, EJ and HJ Bohnert. Development and Environmental Stress Employ Different Mechanisms in the Expression of a Plant Gene Family. Plant Cell, 1993 November. 3. DeRocher, EJ and F Quigly, et al. The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum, a facultative CAM plant. Molecular Genetics, 1993 June. 450-62. 4. Dhingra, Amit and Archie R. Portis, Jr., et al. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Plant Physiology, April 20, 2004; 101(16): 6315-6320.
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