Results (view as seperate page)
Figure 1. Bradford Assay: Correlation between the concentration of protein
samples and absorbance at 750nm.
| Tray | Date Planted | Harvest Date | Description |
| 1 | 6/17/2005 | 9/5/2005 | Senescing, Seed Producing |
| 2 | 6/24/2005 | 9/6/2005 | Bolted, Flowering |
| 3 | 7/8/2005 | 9/7/2005 | Bolting, No Flowers |
| 4 | 8/5/2005 | 9/8/2005 | Young plants, Leaves only |
Table 1. Sample Descriptions by Tray.
| Well | Dye | Type | Label | C(T) | Color Code |
|---|---|---|---|---|---|
| A7 | Run1 | SBG1 | 100ng Sample from Tray 1 | 12.181 | Thick Red |
| A8 | Run1 | SBG1 | 150ng Sample from Tray 1 | 14.062 | Cyan |
| C7 | Run1 | SBG1 | 100ng Sample from Tray 3 | 10.973 | Blue |
| C8 | Run1 | SBG1 | 150ng Sample from Tray 3 | 11.892 | Purple |
| E7 | Run1 | SBG1 | 50ng Sample from Tray 1 | 32.021 | Pink |
| E8 | Run1 | SBG1 | Blank | 13.217 | Thin Red |
Table 2. PCR Data from the plate read at 84°C.
Figure 2. PCR using Primer A.
Figure 2 and table 2 show the Ct value of the PCR products.
The 100ng sample 3 product had the smalled Ct value (10.97) and the 50ng sample 1 product had the largest ct value (32.02). The blank also crossed the threshold line, with a ct value of 13.22.
Figure 3. Agarose gel of PCR products.
Figure 3 shows that on the gel of the PCR products, D8 shows no bands, E8 (blank) shows a weak band, and the other lanes show strong bands at the same point as the molecular weight marker.
| Well | Dye | Type | Label | Tm | Color Code |
|---|---|---|---|---|---|
| A7 | Run1 | SBG1 | 100ng Sample from Tray 1 | 84°C | Left Red |
| A8 | Run1 | SBG1 | 150ng Sample from Tray 1 | 84°C | Green |
| C7 | Run1 | SBG1 | 100ng Sample from Tray 3 | 84.4°C | Blue |
| C8 | Run1 | SBG1 | 150ng Sample from Tray 3 | 84.6°C | Purple |
| E7 | Run1 | SBG1 | 50ng Sample from Tray 1 | 65°C | Orange |
| E8 | Run1 | SBG1 | Blank | 65°C | Right Red |
Table 3. Data from PCR melting curve.
Figure 4. PCR data showing the melting temperatures of the samples.
Table 3 and figure 4 show the melting points of the PCR products. A7, A8, C7 and C8 all had a Tm of about 84C. E7 and E8 both had a Tm of 65C.
| Sample | Abs @ 260nm | Abs @ 260nm | Purity |
| Tray 1 | 0.022 | 0.174 | 0.1264 |
| Tray 2 | 0.013 | 0.062 | 0.2097 |
| Tray 3 | 0.015 | 0.052 | 0.2885 |
| Tray 4 | 0.059 | 0.080 | 0.7375 |
Table 4. Purity of DNA.
Figure 5. SDS PAGE acrylamide gel stained with Comassie Blue.
The comassie blue gel shows samples 3 and 4 having strong bands. Sample 2 having weaker bands. And sample 1 having very weak bands.
Figure 6. Membrane of the western blot.
The membrane from the western blot shows samples 2 and 3 having strong bands similar to the molecular weight marker. Sample 4 has a slightly lighter bands. And sample 1 has no visible bands.
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Introduction (view as seperate page)
Rubisco, or ribulose 1,5-bisphosphate carboxylase, is thought to be the most prolific protein on the face of the earth. It is an enzyme which is used in the Calvin cycle to catalyze the first major step in carbon fixation. About 50 percent of all protein found in leaves is Rubisco and it is the major pathway by which inorganic carbon enters the biosphere. It is this remarkable feat that make Rubisco the start of the food chain.
During photosynthesis Rubisco is active. However, in darkness the enzyme ceases to function. The stroma increases its pH from 7.0 to 8.0 during photosynthesis, which activates the protein. Rubiscos’ narrow optimal pH is centered right around 8.0. When darkness falls Rubisco is stopped by competitive inhibitors synthesized by plants.1 The regulation of Rubisco brings up the interesting question of what other aspects of plant development would affect the Rubisco content. We sought to explore the affects on development and Rubisco expression. Pervious research shows that during development of tobacco leaves the Rubisco expression, measured by CO2-exchange rate, peaks in young plant leaves.2 This indicates that young plants have a great Rubisco content than old leaves. A possible explanation to this would be that once a plant begins to advance towards replication energy is devoted to seed development. As some plants age, a break down of plant tissues can be observed while they are still living. This tissue break down is usually during seed development and is termed senescing.3 Senescing could be why Rubisco content varies between plants. In the following experiment we will track the development of Arabidopsis thaliana plants from seedlings to reproductive adults and measure the Rubisco content in their leaves. |
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Discussion (view as seperate page) We hypothesized that older plants would have less Rubisco expression than younger plants. This was based on the action of senescing. As plants age and begin seed production energy is devoted to perpetuating the species. Thus, Rubisco expression falls as it becomes secondary. Arabidopsis thaliana was chosen for study because it is an annual plant, the life cycle is relatively short, and there was a high availability through the biology department. Leaves were harvested at random from four different sets of plants, termed trays one through four. Tray one held the oldest plants which were flowering and had obviously senesced leaves. Trays two and three held plants which were bolting, but only tray two had begun to flower. Tray four held young plants which were a month old and had developed into leafy stubs. Real-time PCR was conducted to determine the level of gene expression by analyzing Rubisco DNA. Samples of 50ng, 100ng, and 150ng were analyzed using primer A. We selected primer A because it was tried and true for RT PCR. Ct values were compared for each tray of plants. The RT PCR data from our experiments became splotchy and made it difficult to identify trends. The Ct values in figure 2 are only present for trays one and three. This is most likely due bad technique and not because of a lack of Rubisco coding DNA in the leaves. Table 4 indicate that most of the DNA we extracted had a decent purity (A260/A280 was close to 1.8) except for that of tray 4, which had a large amount of impurities giving it a ration of .7375. The Ct values for tray three are 100ng at 10.973 and 150ng at 11.892. These are clearly less than those for tray one, 100ng at 12.181 and 150ng tray 14.062. The Ct values are most likely greater for 100ng samples because the 150ng samples were two concentrated and repressed the replication process. A lower Ct value corresponds to a larger concentration of DNA. These values indicate that older seed producing plants have less Rubisco coding DNA in their leaves than maturing plants. We must also note that our blank had a Ct value of 13.217 (red line), which is the second quickest Ct value in our data. This is obviously a result of poor technique when establishing our PCR samples. Further exploration of our PCR data using an agrose gel is shown as a figure 3, stained with sybr green . We observed that all of the samples which had Ct values contained DNA. Most notably we can see that the blank, lane E8, contained Rubisco coding DNA. Obviously, Rubisco coding DNA found in the blank sample indicates that our results should be taken with a grain of salt. We also considered the expression of Rubisco by measuring the concentration of its large subunit in plant leaves using several methods. Our Western Blot, shown in figure 5, indicates the concentration of Rubisco large subunits, which appear around 47,600Da. Trays one and two have a weak concentration of the large subunit. Trays three and four indicate a substantial concentration of protein. Thus, tray one (older plants) having more Rubisco coding DNA than tray four (young plants). Protein is only produced after transcription and translation. This gives two possibilities of why older plants contain more large Rubisco subunits. It could either be that older plants just have more Rubisco coding DNA and a higher expression or that young plants have a greater level of expression, but just have not processed the DNA into protein. Our Western Blot transfer membrane is displayed in figure 6. The data displayed here is consistent with that of the Western blot. The photo taken with a BioRAD Chemilumenescent camera reveals protein in trays two through four. Tray one appears to have no large Rubisco subunits in it at all. Again, this shows that developing plants contain more Rubisco protein than seed producing, senescing plants. Our results for DNA and Rubisco protein analysis seem to be contradictory to one another, but our original hypothesis may hold true. The fact that our blank sample in RT PCR contained Rubisco coding DNA combined with a low DNA purity for tray four leads note that these test may be unreliable. However, more Rubisco subunits found in older plants does not necessarily indicate that their expression for the protein is greater. As mentioned before DNA must be converted to protein and it must also be noted that the degradation of Rubisco is not factored into our results. Our PCR data indicates that younger plants do contain more Rubisco coding DNA. We propose that younger plants do have a greater Rubisco expression based on this and the reason we see more Rubisco subunits in older plants is due to them having previously produced protein. |
Methods (view as seperate page)
Protein Isolation and Analysis
http://csm.jmu.edu/biology/.../protein_isolation.htm
The first step was to isolate the rubisco proteins from grounded Arabidopsis. Four samples were harvested, varying from very young to old, flowered senescing plants. The plants samples used were 34, 61, 74, and 80 days old. After centrifuging to separate the protein from DNA and other substances, some of the protein was put into the -80°C freezer. The rest was used for a DC assay.
The DC assay contained no .0625 mg/ml standard as the protocol called for. Reagent B and SA were added to reduce Cu2+ to Cu+ and then using the Cu+ and the aromatic amino acids present in the sample to reduce the Folin-Ciocalteu reagent to its blue colored species (red light absorbing). Absorbance was measured at 750nm and the concentration of protein was determined using Beers Law.
Reagent S: detergent used to break down cell and help extract protein
Reagent A: Cu2+, reduces to Cu+ based on amount of protein present in sample
Reagent B: Folin-Ciocalteu reagent, reacts with sample to create a species that absorbs in the red spectrum, away from pigments in plant.
Reaction 1: Copper is reduced based on amount of Rubisco in sample, this Cu+ is itself a blue light absorbing species that can be monitored however there is potential for interference from pigments present in sample.
Reaction 2: The sample is reduced again this time to a blue sample that is more strongly absorbent but, in the red range of the spectrum, which won’t interfere with pigments in the plant.
Western Blot Procedure
http://csm.jmu.edu/biology/.../westernblot.htm
The isolated protein was then run on a Western Blot. The first step was to do an SDS PAGE, where the proteins separated on the polyacrylamide gel based on size. The PAGE was run for two gels, one to be stained and the other electrobloted. After electrophoresis, one gel was submerged in Comassie blue stain which stuck to aromatic amino acids in the proteins. Using the BioRad imager, a picture of the stained gel was taken showing the size of the proteins isolated in comparison to the molecular weight marker. The rubisco proteins would show up at the 53,000 marker.
The second gel was pressed with a membrane to transfer the proteins onto it. After overnight transfer from the gel to the membrane, the membrane was stored in blocking buffer until the next procedure.
Antibody Detection
http://csm.jmu.edu/biology/.../antibody%20detection.htm
The final step with the proteins was to use antibody detection to find the amount of protein on the membrane. We used the alternative light reaction procedure. A chicken antibody was used to bind to the rubisco proteins on the membrane, followed by a rabbit anti-chicken antibody which was linked to the enzyme horse radish peroxidase, and enzyme which gives off light. With these antibodies stacked onto the Rubisco proteins, the membrane was put into the Biorad camera to measure the amount of light given off by the horse radish peroxidase.
Genomic DNA Purification from Plant Sources
http://csm.jmu.edu/biology/.../plantdna2003.htm
The second portion of the experiment involved isolating DNA and determining the amount it that coded for Rubisco. Just like in isolating the protein, Arabidopsis was harvested and ground down. This time Chloroform and isoamyl alcohol were added to sample. The chloroform was used to bind all proteins and polysaccharides present in the sample, this mixture was then centrifuged out and became the bottom fraction in our sample. The DNA and tris-buffer solution was the supernatant that was drawn off after centrifugation. Isopropanol was added and the sample was again centrifuged, supernatant discarded, and ammonium acetate was added the precipitant and then centrifuged down to a pellet.
The DNA concentration was then determined by spectrophotometry at 260nm and 280nm, and beers law was used to determine the concentration from the measured absorbance.
Real Time PCR
http://csm.jmu.edu/biology/.../pcr2004.htm
The next step was to find if the Rubisco gene was present in the plant samples using a Polymerase Chain Reaction. After finding that Primer set A would work on Arabidopsis, it was selected to cut a fragment of DNA about 500bp in size. Primer A consisted of a forward primer of rbcl2f and a reverse primer of RBCL-fonfana.
The available PCR tubes were filled with 50ng, 100ng, 150ng samples of each of the four samples, and one blank. The concentrations were set up like this:
Agarose Gel Electrophoresis
http://csm.jmu.edu/biology/.../agarose2003.html
The final objective was to run the PCR DNA fragments on Agarose Gel in order to separate out the products and determine how much Rubisco was present using a 1kb plus ladder. A 2% agarose gel was used because it resolves for 100bp to 3kb fragments, and the DNA fragments created by the PCR were 500bp in size. A UV photo was taken of the dyed gel when electrophoresis was completed.