Results

To start the experiment, abbreviations were given to each tissue sample. The stems, leaves and petals from Chrysanthemum were abbreviated CS, CL, and CP, respectively. The stems, leaves and petals from Dianthus were abbreviated DS, DL, and DP, respectively.

             Leaf, petal and stem tissue were cut and ground in order to begin protein isolation. Once proteins were isolated, protein concentration for each tissue sample was calculated using spectrometry. Samples were diluted with QB Buffer and mixes with Reagent SA and B. After 15 minutes, the sample's absorbance was measured at ABS 750. Results are shown in Table 1. Absorbance was also measured for standard’s concentration shown in Table 2.  

           Table 1. Diluted samples of plant tissues and their corresponding absorbance at ABS 750. The highlighted dilutions were used to further the experiment since they fell on standard curve see in Figure 1. 

                                 Table 2. Protein standard’s concentration and their corresponding absorbance at ABS 750.

A standard curve was created using our standard’s concentration and absorbance data shown in Figure 1. The equation of the line was found to be y = 4.7709x + 0.0447. The R² value was also found to be 0.9498. When comparing the standard curve to the absorbances of the diluted samples, the 1:20 dilutions of each sample fell closest on the standard curve. Using the equation of the line in Figure 1, protein concentrations of each sample was calculated. By inserting the 1:20 absorbance for each sample in the “x” of the equation, a “y” value was calculated. Multiplying the “y” value by the dilution factor of 20 gives the concentration of each sample. The calculated concentration of each sample is shown in Table 3.

          Figure 1. Graph of protein standard's concentration versus their absorbance at 750nm. A linear relationship was anticipated since increased protein concentration is proportional to increased absorbance.

                                 Table 3. Concentration of each tissue samples calculated by using the line of the equation and absorbance of each tissue sample's 1:20 dilution.

SDS Page and Western Blot Analysis

 An SDS gel electrophoresis was setup in order to separate each sample’s proteins by their charge and size. The larger subunit of the Rubisco protein was also loaded in one lane as a positive control for the procedure. A rainbow marker was also loaded as a standard. 30ug of protein from each tissue sample was loaded into each lane and the gel was stained with Comassie Blue. SDS gel electrophoresis results are shown in Figure 2. The larger subunit of the Rubisco protein was not found in any of the lanes of the Native SDS gel electrophoresis. If Rubisco was shown, its band would have been between the green (85kDa) and violet lanes (45.7 kDa) of the rainbow marker. Rubisco was not found in any lanes. The Green marker was detected. The violet marker was also not seen in the gel and therefore cannot be marked in Figure 2.   

Figure 2. SDS gel electrophoresis. Lane 1- CS, Lane 2-CL, Lane 3-CP, Lane 4-Rainbow marker, Lane 5-Rubisco Standard, Lane 6- DS, Lane 7-DL, Lane 8-DP. Rubisco protein was not located in any of the lanes. Green marker in Rainbow marker was located.

       A Western Blot was perforemed in order to detect the larger subunit of Rubisco in tissue samples using antibody detection. The primary antibodies were made from chicken and has a conserved region of the large subunit of the Rubsico protein. The secondary antibodies were made from goat that would bind to the chicken antibodies. An luminescent enzyme is linked to the secondary antibody in order to see results. 30ug of protein from each tissue sample was loaded into each lane and the gel was then electroblotted. Rubisco protein was detected in lanes for CS, Positive Marker, DS, and DL. The darkest band out of the ones detected was the band for CS. The band for DS was darker than the one for DL. Rubisco protein was not detected in lanes for CL, CP, and DP. 

                         Figure 3. Results of Western Blot. The larger subunit of the Rubisco protein was detected in Lanes 1,5,6 and 7. Lane 1-CS, Lane 2-CL, Lane 3- CP, Lane 4-Rainbow marker, Lane 5-Positive Control, Lane 6-DS, Lane 7-DL, Lane 8-DP.

              Leaf, petal and stem tissue were cut and ground in order to begin DNA isolation. After DNA was extracted from the plant source, the DNA samples were diluted in order to get an absorbance reading between 0.1 and 1. After the dilution, the absorbance at 260nm and 280nm were determined for each of the plant samples. Purity of the samples was then calculated by dividing ^Abs260/_Abs280. The concentration of each DNA sample was calculated by the equation: ^µg/_µl = Abs260 * ^50µg/_µl * dilution factor of 28.56. Table 4 contains for results of DNA absorbance and concentration.

  Table 4. DNA absorbance reading at Abs260 and Abs280 after extraction and dilution. Purity of DNA and DNA concentration for each of the plant's sample was calculated. If purity value is above 1.83, samples contain little protein.    

           Real-Time PCR was performed in order to determine which samples had the highest concentration of Rubisco DNA. The primers rbcl2F and RBCL-Savolainen were used in this experiment. Using these primers would result in a 200bp DNA fragment. Cyber Green dyes were used in the PCR in order to detect fluorescence. The lower the C(T) values, the more DNA was present in that sample. Each sample and blank was placed in two wells for a total of 14 wells in this experiment. Therefore each sample will have two different PCR trial results. All plant tissue samples, except DP and the blank, had similar Tm.  Results of the PCR are shown in Figures 4-9 and Tables 5.

Figures 4-5, Table 5. The location of tissue samples and blank along with their corresponding wells during Real-Time PCR and C(T) values for each of the samples and blank.

Figures 6-7. Tm of plant tissue products in Real-Time PCR. Tm is the defined point when 50% of primers have denatured and 50% have bound to template. Curves of graph corresponds to fluorescence intensities in tissue sample. For Trial 1, a Tm around 82-83º is shown for all samples, except G9 and E9. For Trial 2, A Tm around 83-84º is shown for all samples, except G10 and E10

Figures 8-9. Graph of fluorescence versus cycle number in Real-Time PCR. Curves of graph correspond to fluorescence intensities in tissue samples. C(T) values are the cycle number as to when the samples reaches threshold (dashed line). Lower C(T) value corresponds to higher concentration of DNA in sample. For C(T) values for both trials, see Table 5.                                     

                 An agarose gel electrophoresis was used to separate DNA fragments which were generated by the Real-Time PCR. Using the primers rbcl2F and RBCL-Savolainen, 200bp fragments should appear in the gel electrophoresis. Figure 10 shows results of the electrophoresis. Results from the gel electrophoresis of the Rubisco DNA showed that 200 bp Rubisco DNA was found in lanes for CL, DL, and CP. The brightest bands of DNA were found to be in the lanes for DL and CP and the lightest band to be in the CL lane. A faint band of Rubisco DNA was found in the “No DNA” lane probably due to contamination during methods of this experiment.   

                               Figure 10. Gel electrophoresis of DNA fragments of PCR tissue samples, blank, and 100bp ladder. 200 bp Rubisco DNA was found in Lanes 1, 2, 4 and 5.  Lane 1- CL, Lane 2- DL, Lane 3-ladder, Lane 4-No DNA, Lane 5-CP, Lane 6-DP.

*Note: Abbreviations were given for each of the tissue samples.

CS- Chrysanthemum Stems

DS- Dianthus Stems

DL- Dianthus Leaves

DP- Dianthus Petals

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