Discussion
Rubisco is essential to the survival mechanisms in plants. It was hypothesized that various parts of Colocasia esculentum (elephant ear) would contain different amounts of Rubisco. Since the leaves receive the most light and most photosynthetic activity occurs there, it was hypothesized that they would contain the most Rubisco followed by the stem and root in protein amounts. Using various techniques as mentioned in the methods, conclusions were made about the above hypothesis. Colocasia esculentum was used because it a fairly common plant and would yield a necessary sample size for testing.
The protein was extracted and isolated from each region of the same plant. From calculations obtained through the DC assay standards, it was found that the leaves had the highest amount of isolated protein followed by the stem and root. This does not reflect the amount of protein present in the plant as a whole. Instead, it is only applicable to the amount that we were able to isolate. Based on the type of tissue, each has varying characteristics that make the sample more or less likely to be able to isolate DNA or protein. The protein isolated in the sample was not necessarily Rubisco, so through further experimentation it was determined that Rubisco was present.
Western Blot Analysis was performed using electro blot and staining techniques. The results from the Western Blot were positive support for the presence of Rubisco in most samples. The electro-blot membrane photo revealed a dark band at approximately 53 kb, which is the size of Rubisco, for the leaf sample; unfortunately, there was an extremely faint band at molecular weight for the stem sample and no band for the root sample. What the membrane did not display the stained acrylamide gel did reveal. The presence of Rubisco was seen on the stained gel for all three samples with the heaviest band for the leaf sample. Though the band for the root sample is not so clear there is still a faint band there. This may indicate the presence of a small amount of Rubisco for the root sample, but this test alone is not sufficient to make that conclusion. Rubisco was in fact present in the leaf and stem samples. The faint band for the root sample may have been due to the small amount of Rubisco present or not loading enough protein for the root. The antibody complexes may have been washed away after hybridization or the lack of color produced by the bands. Something that could have been done was to increase the protein concentration for that sample. We should have had a consistent amount of 30ug of protein for each sample. However, since this was not present, it was hard to compare the amount of Rubisco in that protein.
The amount of Rubisco was also determined by quantifying the DNA that encodes for the Rubisco protein. The abs value was the highest for the leaf sample, which again could indicate the largest amount of Rubisco based on DNA, but this stage of the experiment was not sufficient to make that assumption. The samples were then run on Real Time PCR and agarose gel PCR. In comparison to the molecular weight, it appears that Rubisco is present for all samples for primer A and primer C. However, between the two trials run for each of the primers, trial 1 showed better results. A very possible reason for this is the pipetting differences between Anita (trial 1) and Lindsey (trial 2).
Running a PCR is advantageous because it shows what size the molecules are in the sample. For the purpose of this experiment, samples thought to contain the DNA sequence for Rubisco were run on a PCR gel. To compliment the agarose gel results, Real Time PCR was also run for the same sample. A Real Time PCR is even more advantageous as opposed to a regular PCR, because of its speed, ability to quantify the amount of protein, and that single nucleotide changes or “mutations” can be identified. The quantified Real Time figures display the cycle number, threshold and amount of fluorescence (Figure 5). The strength of fluorescence indicates the amount of DNA present in the sample. The C(t) values, or point at which the cycle number meets the threshold line, are used to quantify the amount of protein or DNA in a sample. A lower C(t) value indicates more expression of the protein of interest.
According to the Real Time graphs, the stem had the smallest C(t) value, indicating that it contained the highest DNA expression. This did not correlate with the Western Blot results, which suggested there was more Rubisco present in the leaf. As seen on the membrane in Figure 2, there was a dark band at approximately 53kb for the lane containing the leaf sample, but very faint bands for the stem and root sample. This was confirmed by the stained gel, which displayed the heaviest band for the leaf sample at 53kb and light bands for the stem and root. The data from the Western Blot and stained gel were suggestive of a heavier Rubisco content in the leaf. It is probable that there was contamination in samples as suggested by the PCR results for the negatives. The presence of contamination for primers A and C can be seen in the original PCR method results and Real Time results (Figure 4 and Figure 6). Our hypothesis that the leaf contained the highest amount of Rubisco was refuted based on the Real Time PCR results. The only part of our hypothesis confirmed was that the root contained the least amount of Rubisco compared to the leaf and stem.
Primers A and C were used to isolate the DNA sequence that codes for Rubisco. It was pre-determined that primer A would yield a 500 bp DNA fragment, while primer C would yield a 200 bp fragment. It was estimated that primer C would be more precise, since the length of the desired sequence was 200 bp as seen by the PCR acrylamide gel. This assumption was confirmed by the PCR gel, which displayed a heavy band at 200 bp for all three samples, leaf, stem, and root, when compared to the molecular marker and no band at the 500bp level. Real Time results indicated that primer A did in fact yield 500 bp Rubisco DNA sequences for all three samples as well, but the data from primer C were more consistent. In further experimentation, primer C would be the best candidate for Rubisco DNA binding. However, for this experiment, a primer C that was not contaminated with protein or Rubisco would have provided more supportive or refutable results.
The importance of the melting curves is to see what temperature the samples C(t) values were taken and to determine the Tm or the temperature at which the products melt (Figure 6). The negative contains primer dimmers, so it is essential that these be melted off so that it would not interfere with the fluorescent reading of the targeted DNA sequence. The Tm for the primer A reaction was between 79ºC and 81ºC. Due to the contamination of primer C, a Tm cannot be determined for trial 1 or Trial 2 as seen in Figure 6, since the negative overlaps with the sample sequences. This contamination was also displayed for the PCR gel.
It cannot be concluded that light and photosynthesis are the only determining factors of Rubisco content, since the stem receives less light than the leaves of this plant and contains the most Rubisco according to this experiment. More experimentation needs to be conducted to confer or refute the quantity of Rubisco for Colocasia esculentum.
Further research:
-A lab protocol that is more strict when quantifying Rubisco and DNA…another way to ensure that what you have is only Rubisco (more sterile techniques should be used to prevent contamination)
-This hypothesis should still be tested, though conclusion can be made, it is wise to repeat this experiment or a similar one to determine if our hypothesis really was true. Conducting an experiment only once cannot ensure any result, which is why repeat trials need to be done. Various sources of error can be weeded out with more trials.
-use other types of the elephant plant to see if the same conclusion is reached
-relation of chlorophyll and Rubisco amounts... is more chlorophyll indicative of more Rubisco?
Back to the Main Page