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Abstract   Introduction    Materials & Methods    Results    Discussion    Literature cited

Isolation and Characterization of the Rubisco Large Subunit

The DC Protein assay system was used to determine how much of each sample to load in the Western Blot gel.  The protein standards and dilutions of the proteins isolated from the three tissues were measured at 750nm and a standard curve was constructed (Figure 1).  The 1:20 diluted protein from the three plant tissues had absorbance values that fell on the standard curve. The average of the three absorbance values for the white tissue was 0.245 which corresponds to a 1:20 diluted concentration of 0.890 mg/ml and an undiluted concentration of 17.79 mg/ml. The average of the absorbance values for the green tissue was 0.217 which corresponds to a 1:20 diluted concentration of 0.777 mg/ml and an undiluted concentration of 15.55 mg/ml.  The average of the absorbance values for the green leaf margins of the white leaves was .188 which corresponds to a 1:20 diluted concentration of 0.660to mg/ml and an undiluted concentration of 13.19 mg/ml.

Figure 1. The protein standards used to determine how much protein had been isolated from the three plant tissues of S. arboricola. The concentrations of the 1:20 dilutions were 0.245 mg for white, 0.245 mg for green, and 0.188 mg for green/white.

To visualize the proteins isolated from the three tissues 120 µg of protein from each sample was run on a polyacrylamide gel. Protein smears were observed from all tissue samples. At approximately 50 kDa a dark smear was visible in the green and green/white lanes which was not observed in the white lane (Figure 2).   

Figure 2.  A Polyacrylamide Gel stained with comassi blue of the protein isolated from the three leaf portions of Arboricola Schefflera. All three tissues yielded a smear of proteins, but the green leaves and the green leaf margins had dark bands at about 50 kDa which was less distinct in the protein from the white tissue.

            A western blot was used to select for the RuBisCO protein.  An anti-RuBisCO chicken antibody and a secondary goat anti-chicken enzyme were used to select for the RuBisCO large subunit. Bands of equal size were observed in protein extracts from green leaves and the green leaf margins. There were no antibodies bound in the protein from the white plant tissue (Figure 3). 

Figure 3. Western blot analysis performed with proteins extracted from variegated and non-variegated leaves of the Arboricola schefflera. Two bands were visible where antibodies bound to protein isolated from the green leaves and the green leaf margins but no bands were visible in the protein isolated from the white leaf tissue.

Isolation and Quantization of the DNA Encoding the RuBisCO Large subunit

DNA extraction was performed on all the leaf samples but was successful for only the green leaf tissues. Nanodrop analysis of the green tissues revealed a DNA concentration 11.1 ng/ml with an A260/A280 ratio of 0.85. Real time PCR was performed on the extracted DNA from the green tissue, a No DNA control and a set of duplicate standards.

A melting curve analysis of the green samples and the no DNA control indicated that amplification  of the target sequence began around 81ºC . Complete degradation of the DNA occurred by 84ºC. A similar melting curve was observed in the three samples of DNA from the green leaves and from the no DNA control (Figure 4).

Figure 4. The melting curve of the real time PCR amplification of the green samples and a no DNA control. A similar melting curve was observed for all the green tissue samples and the no DNA control.

The C_T values were calculated for all real time PCR samples using a threshold value 0.02. A standard curve was constructed from a plasmid containing the RuBisCO gene to calculate the mass of the RuBisCo DNA from the green leaf tissue (Figure 5).  The accumulation of sybr green dye bound in amplified DNA was used to determine how much starting RuBisCO DNA was present in the three green samples (Figure 6).

Figure 5. The standard curve generated from a serial dilution of a plasmid containing the RuBisCO gene. This data was used to determine the starting mass of the RuBisCO DNA from the three green samples.

Figure 6. The accumulation of sybr green dye in products of the real time PCR of the three green samples and one No DNA control. The No DNA control had more starting DNA than green sample 3.

The C_T value for the first green samples was 17.61 which corresponds to a mass of the starting RuBisCO DNA of 0.02285ng.  The C_T value for the second green sample was 18.253 and had a mass of 0.01739 ng. The final green sample had a C_T value of 21.237 which had a mass of 0.004899 ng . The no DNA control exhibited a C_T value of 18.891 and had a starting DNA mass of 0.01327 ng (Table 1).  

Table 1. The C_T values for the three samples from the green leaf tissue and the no DNA control. The three green leaf tissues had a broad variation in starting mass. The No DNA had a significant starting mass of DNA, larger than green 3.

Agarose Gel of PCR samples

    An agarose gel of the real time PCR samples was run using the green samples and the No DNA control to determine if the correct DNA fragment had been amplified.  No DNA was observed at 200 bp in any of the lanes.  A very small DNA fragment, less than 100 bp, was observed at the bottom of the gel in every lane, likely primer dimer (Figure 6).

Figure 6. An agarose gel stained with ethiduim bromide. No DNA fragments were observed in any of the lanes at 200 bp. very small fragments, less than 100 bp was observed in all the lanes.