Discussion

Real Time PCR

Rubisco is the most prevalent enzyme on the earth and is involved in one of the most important processes of life: photosynthesis. Its role is to fix carbon dioxide into a compound that is used to create sugars that provide plants with energy.  Thus, it is important to understand how environmental factors, such as water requirement, affect genomic and expression levels.

To determine the effect of a plant’s water requirement, samples were taken from plants with varying degrees of water needs.  DNA was purified from samples of a drought resistant plant, purslane, a mesophyte, lavender, and an aqueous species, water lettuce. Reverse primers, rbclf and rbcls, were designed to amplify the RBCL gene encoded in the chloroplast using real time PCR.  DNA standards of known concentrations were also amplified in order to determine the exact concentrations of DNA in our samples.  However, the standards did not run properly, so they were excluded from analysis.  Possible reasons for the poor results from the standards could be due to improper pipetting and contamination.  The melting curve of the samples amplified by rbclf showed a series of peaks all around the same temperature, 85°C, indicating the same piece of DNA was amplified.  There were no other large peaks, suggesting no contamination of other DNA in the samples.  However, the control of no DNA actually had a peak around the same temperature as the other samples which means that the control had some contamination. Thus an agarose gel had to be run to ensure that the amplified sequence was indeed Rubisco and not a mere contamination. The agarose gel did indeed confirm that the sequence amplified was Rubisco because a 500 bp band appeared which is the predicted size of a Rubisco fragment using an rbclf primer.

The melting curve of the samples amplified by rbcls was not nearly as clean.  Several large peaks occurred at varying temperatures.  The no DNA control also showed contamination.  Since the amplification by rbcls was not as a clean as the amplification by rbclf, only the rbclf samples were used to analyze the RBCL content.

Using the melting curve, plate readings were analyzed at  82°C, cycle 15, when all of the contaminates where melted off and right before the target sequence began to melt.  Purslane had the highest average C(t) value at 23.2.  This indicates purlsane had the least amount of DNA because it took 23.2 cycles to reach the threshold value.  Lavender and water lettuce had very similar C(t) values.  Lavender had a value of 16.0 and water lettuce at 17.1. Purslane was predicted to have the lowest amount of DNA since it is a drought resistant plant.  Drought resistant plants must conserve water and close up their stomata.  Oxygen gets trapped in the plant and Rubisco also has a very high affinity for oxygen as well as carbon dioxide.  Since oxygen begins binding to Rubisco, carbon dioxide does not bind as efficiently, thus drought resistant plants have found other enzymes that have lower affinities for oxygen.  Since lavender and water lettuce do not have much problem receiving water, they do not have as much of a problem of oxygen binding.  Therefore they can afford to keep producing lots of Rubisco.  Hence it was expected for both of them to have higher amounts of DNA than purslane.  Yet, it was predicted that water lettuce would have more DNA then lavender when it appeared to have almost the same amount as lavender.  This suggests that there might be a threshold of amount of water availability that makes it advantageous to produce less Rubisco.  Anything above the threshold, like lavender or water lettuce, does not make a difference and therefore, all such plants might have similar amounts of DNA.  Further study of other mesophytes and aqueous species are needed to determine if this is true.

Western Blot

Running the gel to be stained with Comassie Blue was intended to confirm the presence of Rubisco in our samples, but it instead appeared blank.  The Western Blot shows large bands at the tops of the marker, purslane, and water lettuce lanes, indicating that protein was there.  There were no bands farther down the gel for all the other lanes.  This could be due to concentrations too low to be detected or to the protein not running down the gel. There are bands present for the lavender samples, indicating the presence of protein, but without a Rubisco standard to compare against or a molecular marker with which to estimate size, it is difficult to draw any conclusions.  The fact that the antibodies were bound suggests that the protein is, indeed, Rubisco.  The 1:200 dilution of lavender unexpectedly appeared to have darker bands than the 1:20 dilution, indicating more protein in the sample.  Possible reasons for this could be an error in pipetting the samples.

It is unlikely that the lack of results on the Comassie blue gel is due to protein degradation of our samples since protein did show up on the Western Blot.  The molecular marker and Rubisco standard should have shown up after staining regardless of the condition of our samples.  A more plausible explanation is the complications we encountered with our electrophoresis apparatus.  Running buffer leaked from the bottom of the chamber and slowed down the process, causing bowing in the band as it ran.  In future experiments it would be prudent to closely monitor the gel apparatus and possibly to expose the gel overnight to see if that improves the quality of the picture. 

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