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Abstract

Introduction

Methods

Results

Discussion

Literature Cited

Results

Protein Isolation and Analysis

After attempting to isolate the RBCL protein, we measured the concentration via spectrophotometric readings at 750 nm in order to detect how much Folin-Ciocalteu reagent was reduced in the reaction.    The assay was carried out at this higher wavelength (usually done at 660 nm) to avoid confusion with the chlorophyll absorbance.  Standard protein samples of known concentration were used to create a standard protein concentration curve in ug/uL, Figure 3, with a high correlation coefficient (R²) value of 0.99.  The equation for the line from Figure 3 was used to obtain the protein concentration of each isolated plant protein sample (Table 1). 

• Protein concentration = 4.2932 x Abs_750nm – 0.2907. 

Figure 3.  Standard protein curve of absorbance read at 750 nm vs. protein concentration in ug/uL.  The protein samples were dissolved in QB buffer and mixed with reagent SA and reagent B for 15 minutes.  Microsoft excel was used to generate the linear equation.

Table 1.  Absorbance of each plant protein sample at 750 nm and corresponding protein concentrations using the linear relationship for concentration and absorbance from Figure 1 in ug/uL.  Protein samples were mixed with reagent SA and reagent B for 15 minutes.

SDS-PAGE of each plant protein sample was conducted and stained in Comassie Blue (Figure 4) with no viable results.   No clear visible bands were observed for any plant protein sample in the resulting gel.  The only bands that appeared were from lanes containing the protein ladder and positive RBCL standard.  Two bands are visibly apparent in each of the two lanes at equal distances from the wells.   However, since the ladder did not show any bands the sizes of protein in these bands cannot be determined.

Figure 4.  SDS-PAGE of each plant protein sample.  The gel was stained in Comassie Blue for about 15 minutes with gentle shaking, de-stained in water for one hour and again overnight.  The picture was taken with epi white light on a white surface.

A western blot of the SDS-PAGE also presented poor results (Figure 5).  The only apparent bands are from the positive RBCL standard; however, these bands are not discrete.  With some focus a very light band can be seen in the large leaf sample lane.

Figure 5.  Western blot of proteins from SDS-PAGE (Figure 4) on Immobilon-PDVF transfer membrane.  Western blot was run at 250 mA overnight.  I_gY chicken anti-Rubisco large subunit primary antibody and I_gG goat anti-chicken I_gY secondary antibody were used.  Horse radish peroxidase was attached to the secondary antibody and a staining solution of luminol/enhancer and peroxide buffer was used.  The blot was subjected to chemiluminescence for 45 minutes.

DNA isolation and analysis

Once DNA was isolated from each plant sample, its concentration and purity were determined, Table 2.  Each plant's DNA sample had a favorable purity; the lowest value was 1.86 and highest was 2.8.   Pure samples are considered to be between 1.8 and 2.1. 

Table 2.  DNA concentration in ug/uL and DNA purity based on A₂₆₀ and A₂₈₀ of plant samples.   Quartz cuvettes (1cm) were used.   The extinction coefficient used for DNA to obtain the DNA concentration is 0.050 ug/uL.  The concentration was found by the equation:

[DNA] = A₂₆₀ x 0.050 ug/uL x dilution factor.

The DNA samples isolated earlier were run on real time PCR in an attempt to amplify the Rubisco DNA fragment.  We chose to use the primers RBCL2F (forward) and RBCL-fonfana (reverse), which flanked a larger portion of the sequence than the other primers available. Two trials were conducted and for each trial a quantitation graph, Figures 7 and 8, and a melting curve graph, Figures 9 and 10, was generated.  A standard quantitation graph was also created using standard Rubisco DNA concentrations in real time PCR, Table 3.  The C_t value for each standard seems to have increased as the concentration of the standard increased, except for the 68.3 pg standard.  No C_t value was obtained for any of the plant DNA samples in either trial.  The only samples that passed the threshold value were either the standard 0 ng DNA or the no DNA control.  The melting curves are incomprehensible.  The only distinct peak is in Figure 9 for the standard containing 0 ng of DNA.  This could be due to background fluorescence only, though.

Table 3.  Cycle threshold (C_t) value for each standard Rubisco DNA sample from the quantitation graph for the real time PCR.  RBCL2F forward primer and RBCL-fonfana reverse primer were used.

Figure 6.  Plot of DNA concentration vs.  cycle threshold value (C_t) for the standard samples.  The equation displayed can be used in conjunction with C_t values obtained for our samples during real time-PCR, in which the C_t value would be substituted in for y and the equation would be solved for x, the DNA concentration.

Figure 7.  One of two quantitation graphs of real time PCR of the plant DNA samples.  Only two samples crossed the threshold, however both of them were not supposed to contain any Rubisco DNA.  The gold curve was the standard DNA containing 0 ng of DNA.  The light blue curve was the negative DNA control.  Each plant DNA sample contained 50 ng of DNA.  RBCL12f and RBCL-fonfana forward and reverse primers, respectively, and SYBR green dye were used.

Figure 8.  Second of two quantitation graphs of real time PCR of the plant DNA samples.  Only one sample crossed the threshold, however this sample was not relevant to this study.  The sample belonged to the fern group and was their negative DNA control.  Each plant DNA sample contained 50 ng of DNA.  RBCL12f and RBCL-fonfana forward and reverse primers, respectively, and SYBR green dye were used.

Figure 9.  Melting curve for first of two quantitation graphs of real time PCR.  The melting temperature for the standard DNA containing 0 ng of DNA is visible at about 86^oC.  No other distinguishable melting temperatures are clearly visible. 

Figure 10.  Melting curve for second quantitation graph of real time PCR.  No distinguishable melting temperatures are visible.

An ethidium bromide stained 2% agarose gel of the real time PCR products for each plant sample contained no distinct bands (Figure 11).  Each of the plant sample lanes seems to have been completely covered in the stain.  The 100 bp DNA ladder, in lane 1, was the only sample to produce visible discrete bands.  The negative control in lane 2 has slightly visible bands. This suggests that there was some DNA contamination in the control, and possibly the other reactions as well.

Figure 11.  A 2% agarose gel of PCR generated DNA fragments from each plant sample.  A 100 bp ladder was used in lane 1.  The gel was stained in ethidium bromide and de-stained in water for about five minutes.