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Results

Protein extracts from green, red and brown leaves (figure 1) were quantified by a DC protein assay. Standards (figure 2) showed linear pattern and could be used to quantify protein.  Extract of green leaves had 22.31 mg/mL of protein, extract of red leaves had 11.72 mg/mL of protein and extract of brown leaves had 9.99 mg/mL of protein. 

Figure 1:  Plant material utilized for protein and DNA extraction. Typical leaves used to extract both protein and DNA samples.  From Quercus rubra (red oak).  Green leaves (on left) are live and photosynthetic leaves.  Red leaves (middle) are senescent but still attached by the petiole.  Brown leaves (on right) are senescent and detached before they were collected.

 

Figure 2:  Standard curve for DC protein assay.  Assay performed to quantify protein in extracts from green, red and brown leaves.  Standards with R2 value of 0.9728 high enough to accept and continue quantification.

 

Extracts were run on an acrylamide gels. 300 mg of protein were loaded in each lane. Comassie stained gel (figure 3) shows green leaf extracts to have more full length proteins, including a band at 55 kDa, likely representing RuBisCo large subunit.  Red and brown leaf extracts do not appear to contain any full length protein detectable by comassie staining.

Figure 3:  Acrylamide gel, stained with comassie blue, of protein extracts from Quercus rubra.  Leaf extracts from green, red, and brown leaves were loaded onto a Tris-HCl acrylamide gel.  Molecular weight marker (MWM) is Bio-Rad kaleidoscope prestained standards.  Few if any full length proteins detected in red and brown leaves.  Green leaves have a detectable band at 55 kDa likely representing RuBisCo large subunit. Imaged with Quality One, Chemidoc XRS camera.

 

The second acrylamide gel was electroblotted onto an immobilon-PDVF membrane.  Primary antibody (chicken anti-RuBisCo) then secondary antibody (goat anti-chicken) were hybridized to the membrane and RuBisCo is seen.  A clear band is visible in the green lane where the large band was seen on the comassie gel, around 55 kDa, confirming the band to be RuBisCo large subunit (figure 4).  The Western blot did not detect any RuBisCo in the red or brown extracts.

 

Figure 4:  Western blot for RuBisCo large subunit. Protein from acrylamide gel was electroblotted onto an immobilon-PDVF membrane.  RuBisCo was detected by chicken anti-RuBisCo primary antibody and goat anti-chicken secondary antibody.   The only definitive band seen is in the green leaf lane around 55 kDa; matching the band seen on the comassie stained gel. Imaged with Quality One, Chemidoc XRS camera.

 

The amount of the RuBisCo large subunit gene from DNA extracts of green, red and brown leaves was quantified by using real time PCR.  DNA extracts were nanodropped at 260nm and 280nm to determine concentration and purity.  The quotient on the two readings (A260/A280) was used to determine the purity of the extracts (table 1).  Pure DNA gives a value from 1.8 to 2.0.  Our green, red and brown samples had values of 1.51, 1.48 and 1.36 respectively, indicating each sample was not pure.  Concentrations of green, red and brown extracts were 189.2 mg/mL, 156.2 mg/mL and 256.2 mg/mL respectively.

 

Table 1:  DNA extracts – purity levels and concentrations.  DNA extracts form green, red and brown leaves of Quercus rubra were nanodropped to quantify DNA concentration and measure their purity.  Absorbance readings were taken at 260nm and 280nm.  The quotient of these readings will indicate the purity of the sample (pure DNA will give a value between 1.8 and 2.0).  A260 readings were also used to calculate concentration using Beer’s Law.

DNA Sample

A260/A280 (purity)

Concentration (mg/mL)

Green

1.51

189.2

Red

1.48

165.2

Brown

1.36

256.2

 

RuBisCo large subunit DNA was quantified using real time PCR.  RuBisCo standards in dilutions of 10 were also amplified to determine concentration (figure 5).  Standards fit well except for the 0.01ng standard which did not amplify correctly.  After discarding the data point, the R2 value of the line of best fit was 0.998.

 

Figure 5:  a) Standard curve of RuBisCo amplified with our primers and conditions.  All standards amplified correctly except 0.01ng which was discarded.  An accurate R2 value of 0.998 was achieved after this data point was deleted.  b) Real time PCR data from RuBisCo standards. All standards are confirmed to have amplified correctly except 0.01ng which amplified incorrectly.  CT cycle value is determined when the sample crosses the threshold point indicated by the dashed line.

 

Using our standard curve CT values for our samples could be determined.  Melting curves were analyzed to choose the best temperature plate reading (figure 6).  Our DNA product denatures around 82oC so the plate read at 80oC was chosen. This prevents error due to primer dimers and other impurities.  Of our samples only the 50ng green extract amplified as well as our water negative control (figure 7).  The CT values for the 50ng Green and water negative control were 30.511 cycles and 20.255 cycles respectively.

 

Figure 6:  Melting Curve of amplified samples.  Only samples that amplified – 50ng green (green line) and the water negative control (red line).  Graph shows derivative of the fluorescence vs. temperature plot because slope changes dramatically when product begins to denature.  The tall peaks indicate when the samples denatured.  Our samples started to melt around 82oC so the plate read at 80oC was chosen.

 

Figure 7:  Real Time PCR data for our samples and the corresponding positions on the microtile plate.  Top graph shows our real time PCR data.  Only two samples amplified.  The blue line is the green 50ng sample, and it had a CT value of 30.511 cycles.  The red line is the water negative control that was contaminated, which had a CT value of 20.255 cycles. CT values were determined when the fluorescent values crossed the threshold point indicated by the dashed line.

 

Real time samples were run on a 2% agarose gel to confirm size of PCR product (figure 8).  Green 50ng sample (lane 2) and water negative control (lane 3) were confirmed to have a product of proper size for the primers (~200 bp).  Red 50ng and brown 50ng were run in lanes 4 and 5 respectively and were confirmed not to contain the amplified product.

Figure 8:  2% agarose gel of real time PCR products.  Lane one contains 100bp marker. Lanes 2 and 3 contain Green 50ng sample and the water negative control respectively.  Both lanes show a band at 200bp confirming the real time PCR product. Lane 4 contains red 50ng and lane 5 contains brown 50ng, and neither show product bands confirming they did not amplify.